Novel compounds and methods for inhibiting cell death

ABSTRACT

This invention provides novel compounds and methods for promoting cell survival and/or plasticity, especially in neuronal cells, by targeting the microtubule End Binding (EB) proteins and other associated proteins (e.g., drebrin). Methods for identifying potential modulators of cell death/plasticity are also described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/647,661, filed May 16, 2012, the contents of which are herebyincorporated by reference in the entirety for all purposes.

BACKGROUND

The plus ends of growing microtubules (MTs) accumulate a diverse groupof MT-associated proteins including the end-binding (EB) protein family.Like other MT plus-end tracking proteins (+TIPs), EB proteins mediateinteractions between the ends of MTs, organelles, and protein complexesas well as altering MT stability. Of the three EB family members, EB3 ispreferentially expressed in the CNS and is used to track MT dynamics.EB1 was shown to interact with a conserved binding site in +TIPS—namely,SxIP. EB3 is associated with cellular differentiation, and it may form adimer with EB1 and act also in neuroprotection.

Some recent reports have shown EB3 interaction with PSD-95 at the levelof the dendritic modeling and plasticity. See, e.g., Sweet et al.,Bioarchitecture 2011, 1(2):69-73; Sweet et al., J. Neurosci. 2011,31(30):1038-1047. Thus, EB3 plus-end decorated MTs control actindynamics and regulate spine morphology and synaptic plasticity, throughinteraction with PSD-95, and NMDA receptor activation.

BRIEF SUMMARY OF THE INVENTION

This invention relates to novel compounds, e.g., peptides, that possessbiological activities including promoting cell survival and/orinhibiting cell death upon exposure to a toxic agent, or promoting cellplasticity, especially in neuronal cells, e.g., protecting synapticvitality against synaptic disruption and death. The present inventionalso illustrates for the first time a mechanism of action in protectionagainst cell death: the microtubule End Binding (EB) proteins (e.g., EB3protein) play an active role in the process and are therefore potentialtargets for modulating cell susceptibility to apoptosis.

In a first aspect, this invention provides an isolated peptide thatprotects cells from apoptosis, especially in neuronal cells, e.g.,protecting synaptic vitality against synaptic disruption and death. Thispeptide contains a core sequence of (1) SKIP (Ser-Lys-Ile-Pro, SEQ IDNO:6); (2) SGIP (Ser-Gly-Ile-Pro, SEQ ID NO:7); (3) SRIP(Ser-Arg-Ile-Pro, SEQ ID NO:8); or (4) a core sequence of NAPVSxIPQ (SEQID NO:1, for example NAPVSGIPQ, SEQ ID NO:5) or a conservativelymodified variant thereof (e.g., NAPVTxIPQ), and it has up to 40 aminoacids at either or both of its N-terminus and the C-terminus. Further,the peptide may contain one or more lipohylic moieties. The peptide hasthe biological activity of inhibiting cell death, especially in neuronalcells. In some cases, the core amino acid sequence is NAPVSKIPQ (SEQ IDNO:2). In some cases, the peptide has up to 20 amino acids at either orboth of the N-terminus and the C-terminus. In other cases, the peptideconsists of the core amino acid sequence, such as SKIP (SEQ ID NO:6),SGIP (SEQ ID NO:7), SRIP (SEQ ID NO:8), or SEQ ID NO:2 or 5. In someembodiments, the peptide is modified at one or more locations, forexample, the peptide may be acetylated at the N-terminus or at aninternal K residue, or it may be lipidated at the N-terminus, or it maybe amidated at the C-terminus. In some embodiments, the core amino acidsequence comprises at least one D-amino acid, and may have all D-aminoacids. One example is all D-amino acid SKIP (SEQ ID NO:6), and anotherexample is acetyl-SKIP-NH₂. In one example, the peptide is notNAPVSRIPQ.

In a second aspect, this invention provides a method for identifyingmodulators that promote cell survival or cell plasticity by detectingbinding between a candidate compound and an EB protein, or by detectingthe ability of a candidate compound to promote the expression of an EBprotein. In one first method for identifying a modulator of cellsurvival, at least these steps are performed: (1) contacting, underconditions permissible for protein-modulator binding, an EB protein witha candidate compound; (2) detecting binding between the EB protein andthe candidate compound; and (3) identifying the candidate compound as amodulator of cell survival or plasticity when binding between the EBprotein and the candidate compound is detected. In a second, cell-basedmethod for identifying a modulator of cell survival or plasticity, atleast these steps are performed: (1) contacting a cell that expresses anEB protein, under conditions permissible for the expression of the EBprotein, with a candidate compound; (2) detecting the expression levelof the EB protein in the cell; and (3) identifying the candidatecompound as a modulator that promotes cell survival or plasticity whenan increased expression level of the EB protein in the cell is detected,and identifying the candidate compound as a modulator that suppressescell survival or plasticity when a decreased expression level of the EBprotein in the cell is detected. In either of these methods, the EBprotein may be an EB1 protein, or an EB2 protein, or an EB3 protein,which may be derived from any suitable species (e.g., a human or rodentversion of the EB protein). In either method, step (1) may furthercomprise providing another protein, for example any one of thoseidentified in Tables 2 and 3 (e.g., a drebrin protein of a suitablespecies) to interact with the EB protein and the candidate compound. Inthe cell-based method, the cell used during the screening process mayexpress both EB1 and EB3 proteins, or it may express an EB protein andanother protein identified in Tables 2 and 3. In some examples, the cellis a neuronal cell, such as a PC12 cell. In some cases, cell plasticityis neuronal plasticity or synaptic plasticity.

In a third aspect, the present invention provides a method for promotingcell survival/plasticity or inhibiting cell death by contacting the cellwith an effective amount of a modulator that promotes cell survival orplasticity. Such a modulator may be of any chemical nature: it may be apeptide described in the first aspect of this invention; it may be acompound that, when administered to a cell in an adequate amount, canincrease EB protein expression; it may be a modulator the promotes cellsurvival/plasticity as identified by the screening method described inthe second aspect of this invention. In some cases, the cell is aneuronal cell, which may be present in a patient's body. In some cases,the modulator is a peptide comprising or consisting of the amino acidsequence of (1) SKIP (SEQ ID NO:6); (2) SGIP (SEQ ID NO:7); (3) SRIP(SEQ ID NO:8); or (4) SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5. Themodulators of cell survival/plasticity as described herein or identifiedby the screening methods described herein can be used for treating apatient suffering from a condition involving excessive cell death orinadequate neuronal or synaptic plasticity, e.g., any one ofneurodegenerative disorders, including but not limited to, Alzheimer'sdisease, Parkinson's disease, corticobasal ganglionic degeneration,progressive supranuclear palsy, progressive bulbar palsy, amyotrophiclateral sclerosis, Pick's atrophy, diffuse Lewy body disease, multiplesclerosis, Huntington's disease, a neurodegenerative pathologyassociated with aging, and a pathological change resulting from a focaltrauma (such as stroke, focal ischemia, hypoxic-ischemic encephalopathy,closed head trauma, or direct trauma), peripheral neuropathy, retinalneuronal degeneration (e.g., retinopathy, such as different types ofage-related macular degeneration), or dopamine toxicity. Further, thesemodulators may also be used for treating a patient suffering from acondition involving impaired neuronal plasticity, such as mentaldisorders and neurodevelopmental disorders including but not limited toanxiety disorders (e.g., any one of specific phobias, generalizedanxiety disorder, social anxiety disorder, panic disorder, agoraphobia,obsessive-compulsive disorder, and post-traumatic stress disorder), mooddisorders (e.g., major depression, dysthymia, and bipolar disorder),psychiatric disorders (e.g., schizophrenia, delusional disorder, andschizoaffective disorder), personality disorders (e.g., paranoid,schizoid, and schizotypal personality disorders, antisocial, borderline,histrionic or narcissistic personality disorders; anxious-avoidant,dependent, or obsessive-compulsive personality disorders, and adjustmentdisorders), eating disorders (e.g., anorexia nervosa, bulimia nervosa,exercise bulimia or binge eating disorder), sleep disorders (e.g.,insomnia), sexual or gender identity disorders, impulse controldisorders (e.g., kleptomania and pyromania), substance abuse disorders,dissociative identity disorders (e.g., depersonalization disorder ormultiple personality disorder), developmental disorders (e.g., autismspectrum disorders, oppositional defiant disorder, conduct disorder, andattention deficit hyperactivity disorder or ADHD).

In another aspect, this invention provides a composition that comprises(1) a modulator that promotes cell survival/plasticity as describedherein; and (2) a physiologically/pharmaceutically acceptable excipient.Such a composition is useful for promoting cell survival/plasticity orfor suppressing undesired cell death, especially in neuronal cells,e.g., protecting synaptic vitality against synaptic disruption anddeath, or improving synaptic plasticity. One example is treating apatient suffering from a condition involving excessive cell death orinadequate neuronal or synaptic plasticity, e.g., any one ofneurodegenerative disorders, including but not limited to, Alzheimer'sdisease, Parkinson's disease, corticobasal ganglionic degeneration,progressive supranuclear palsy, progressive bulbar palsy, amyotrophiclateral sclerosis, Pick's atrophy, diffuse Lewy body disease, multiplesclerosis, Huntington's disease, a neurodegenerative pathologyassociated with aging, and a pathological change resulting from a focaltrauma (such as stroke, focal ischemia, hypoxic-ischemic encephalopathy,closed head trauma, or direct trauma), peripheral neuropathy, retinalneuronal degeneration (e.g., retinopathy, such as different types ofage-related macular degeneration), or dopamine toxicity. Another exampleis treating a patient suffering from a condition involving impairedneuronal plasticity, such as mental disorders and neurodevelopmentaldisorders including but not limited to anxiety disorders (e.g., any oneof specific phobias, generalized anxiety disorder, social anxietydisorder, panic disorder, agoraphobia, obsessive-compulsive disorder,and post-traumatic stress disorder), mood disorders (e.g., majordepression, dysthymia, and bipolar disorder), psychiatric disorders(e.g., schizophrenia, delusional disorder, and schizoaffectivedisorder), personality disorders (e.g., paranoid, schizoid, andschizotypal personality disorders, antisocial, borderline, histrionic ornarcissistic personality disorders; anxious-avoidant, dependent, orobsessive-compulsive personality disorders, and adjustment disorders),eating disorders (e.g., anorexia nervosa, bulimia nervosa, exercisebulimia or binge eating disorder), sleep disorders (e.g., insomnia),sexual or gender identity disorders, impulse control disorders (e.g.,kleptomania and pyromania), substance abuse disorders, dissociativeidentity disorders (e.g., depersonalization disorder or multiplepersonality disorder), developmental disorders (e.g., autism spectrumdisorders, oppositional defiant disorder, conduct disorder, andattention deficit hyperactivity disorder or ADHD). A further aspect ofthis invention is a screening method for identifying modulators thatpromote cell survival or cell plasticity by detecting binding between acandidate compound and a target protein, or by detecting the ability ofa candidate compound to promote the expression of a target protein.Instead of using an EB protein as the target protein as described above,any one of the proteins identified in Tables 2 and 3 may be used in thescreening methods in the same manner as described herein. In some cases,any one of these proteins may be used in combination with an EB proteinin the binding assay for identifying a potential modulator. In somecases, two or more of the proteins identified in Tables 2 and 3 may beused together in the screening process, especially in the cell-basedassay format.

An additional aspect of this disclosure relates to compounds that theinventors have identified as possessing biological activities similar tothe peptides described above, e.g., capable of promoting cell survivaland/or plasticity, and/or inhibiting cell death, especially when a toxicagent is exposed to the cells, including neuronal cells. While notintended to be bound to any particular theory of mechanism of action,the inventors believe that these compounds act in a fashion similar tothe peptides described above in exerting their biological activity,e.g., protecting neuronal cells against cytotoxicity. These compoundsare initially identified based on their interaction with the EB proteins(e.g., binding with the EB3 or EB1 protein in an in silico assay). Thesecompounds include: diltiazem, trazodone, acetophenazine, carphenazine,flumazenil, quetiapine, risperidone, fluvoxamine, thiothixene,almotriptan, and methysergide. More information relating to thesecompounds are provided in Table 4 of this application. These compounds,as well as any other compounds that may be identified through the sameor similar screening methods, can be used for promoting cell survivaland/or inhibiting cell death in the same or similar manner that theabove-described peptides may be used according to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: MT plus-end tracking proteins (+TIPs). +TIPs localize to growingMT ends where they form dynamic interaction networks. These networksrely on a limited number of protein modules and linear sequence motifssuch as the calponin homology (CH), EB homology (EBH) andcytoskeleton-associated protein glycine-rich (CAP-Gly) domains, andEEY/F and SxIP sequence motifs (top part). These elements mediate theinteraction with each other and MTs, and typically display affinities inthe low micromolar range. End-binding (EB) proteins are now generallyaccepted to represent core components of +TIP network assemblies as theyautonomously track growing MT plus-ends independently of any bindingpartners. Moreover, EB proteins directly associate with almost all otherknown +TIPs and by doing so target them to growing MT plus-ends. SxIPmotifs act as a general ‘MT tip localization signal’ by interacting withthe EBH domain of EB proteins. Likewise, EEY/F motifs of EB proteins andα-tubulin guide CAP-Gly proteins to MT tips. EBH-SxIP and theCAP-Gly-EEY/F interactions X-ray crystallography analysis (bottom part).The two distinct binding modes revealed by these structures offer amolecular basis for understanding the majority of known interactionnodes in dynamic +TIP networks (adopted from BMR: Dynamic ProteinInteractions, Michel 0. Steinmetz Paul Scherrer Institute).

FIG. 2: The EB1 binding domain. Similar binding domains are indicated inother members of the EB family, the original scheme by the inventors aspresented on the figure was recently corroborated (Laht et al., BiochemBiophys Acta, Feb. 21, 2012).

FIG. 3: EB1, 2, 3 protein sequence alignment. Protein sequence alignmentof EB1,2,3 (MAPRE1,2,3) from mouse, rat and human origin showing highsimilarity. Alignment was done using UniProt ClustalW (Higgins et al.,Methods Enzymol 266:383, 1996). Uniprot identifiers are shown on theleft. Colored rectangular indicate the binding cavity interactingresidues as reported (Honnappa et al., Cell 138:366, 2009).

FIG. 4: EB1, 2, 3 mRNA expressions in cell cultures. Quantitative RNAanalysis (a) Rat cell cultures: rat non-differentiated pheochromocytoma(PC 12) cell line, differentiated PC12 cells treated with NGF andprimary cultures of astrocytes and neurons grown for 4 days in-vitro(4DIV) or 19DIV. siRNA silencing (in the indicated concentration) of EB3in PC12 cells and primary neuronal culture compared to cells treatedwith non-targeting siRNA. (b) Mouse cell cultures: mouse fibroblastsNIH3T3 cell line, non-differentiated (nd) P19 teratocarcinoma cell line,P19 differentiated into neuro-glial-like by retinoic acid (RA) and tocardiomyocyte like cells by DMSO. Neuronal differentiated cellsexhibited a relative high expression of EB3, which is specificallyinhibited by targeted EB3 RNA silencing.

FIG. 5: EB3 binds to NAP. NAP was bound to sulfolink coupling resin andrecombinant EB3 (37Kd) was loaded on the resulting affinity column.Proteins were separated by electrophoresis SDS-PAGE followed westernanalysis. The figure shows the western results. Column loadingmaterial-recombinant EB3 (Load), the column flow through (FT1 and FT2),the column wash (W1 and W2) and the eluted material (E1-4). Each lanewas loaded with 40g1 of sample (including sample buffer): Load=˜11.5 μg,FT1=˜3.1 μg, FT2=˜1 μg, W1=˜0 μg, W2=˜0 μg, E2=˜9 μg. Proteinconcentration was estimated as indicated in the methods section. EB3 wasidentified by specific antibodies.

FIG. 6: EB3 binds SIP and SKIP containing sequences. NAP was bound tosulfolink coupling gel and full length human EB3 recombinant proteintogether with competing peptides were loaded on the resulting affinitycolumn. The figure shows the western analysis results. Column loadingmaterial-recombinant EB3+indicated peptide (Load), the column flowthrough (FT1 and FT2), the column wash (W1 and W2) and the elutedmaterial (E1-3). EB3 was identified by specific antibodies. Left lanewas loaded with protein marker (m). The table lists the sample volumeand estimated protein amount loaded on each lane of the proteinseparation gels.

FIG. 7: NAP protects PC12 cells from zinc intoxication. (a) Zinctreatment (400 μM) resulted in PC12 cell death which was protectedagainst by NAP treatment. Results of mitochondrial activity (MTS cellviability) are shown—100—is 100% survival—control without zinctreatment. (ANOVA, p<0.0001, n=18, post hoc comparison were made inreference to vehicle +Zn treatment). (b) NAP, NAPVSKIPQ (SKIP) andAcetyl—NAPVSKIPQ (Ac-SKIP), (10⁻⁹M) each separately significantlyincreased survival. The protection by the three peptides was similar.(ANOVA, p<0.0001, n=12-30, post hoc comparison were made in reference tovehicle +Zn treatment). (c) Other peptides tested such as NAPVSRIPQ(SRIP) and NAPVTRIPQ (TRIP) were inactive. Results were similar to thecontrol results. Ac-SKIP was used as an active control. (ANOVA,p<0.0001, n=12, post hoc comparison were made in reference to vehicle+Zn treatment).

FIG. 8: Zinc treatment (500 microM) resulted in PC12 cell death whichwas protected against by NAP treatment (10⁻¹⁵M). Several controls wereused one included non-specific siRNA (nci), one (no treatment, cont),and one RNA silencing of EB3 (eb). Results of mitochondrial activity(MTS), (I. Divinski, M. Holtser-Cochav, I. Vulih-Schultzman, R. A.Steingart, I. Gozes, J Neurochem 98, 973 (August, 2006)) areshown—100—is 100% survival—control (n=8 for each experimental point).ANOVA—P<0.001;

Source of Variation DF SS MS F P Between Groups 5 0.526 0.105 6.888<0.001All Pairwise Multiple Comparison Procedures (Student-Newman-KeulsMethod): NAP protects in the control situations, but not in the presenceof EB3 silencing.

FIG. 9: MT in dendritic protrusions. Cortical neurons were treated withvehicle or NAP (10⁻¹²M) at 13 DIV for 2 hrs and then fixed and stained.(a) Representative images showing dendritic protrusion exhibiting Tyr-MT(red) Glu-MT (green) or both. Bars: 10 μm. Bars in magnified areas 1 μm.(b) Representative images showing dendritic protrusion exhibiting Tyr-MT(red) Glu-MT (blue) and PSD-95 (green). Images on the right side are amagnification of the central area of the left side images. Bars: 1 μm.

FIG. 10: Treatment with NAP increases PSD-95 accumulation in dendriticspines. Methods: 1. Primary cultures of neurons were prepared asfollows. Newborn rats were sacrificed on postnatal day 1. Cerebralcortex tissue was then dissected and dissociated individually from eachpup with the Papain Dissociation System (PDS; Worthington BiochemicalCorporation) according to the manufacturer's instructions. 2. Cells werefixed with ice cold methanol, blocked with 2% bovine serum albumin (BSA)and 5% normal goat serum in Tris buffered saline containing tween 20(TBS-T; 20 mM Tris pH 7.5, 136.8 mM NaCl, and 0.05% v/v Tween 20), andincubated with primary antibody followed by the appropriate secondaryantibody. 3. Primary antibodies that were used include: monoclonal antiPSD-95 (ab-2723, Abcam), monoclonal anti Tyr-α-tubulin antibody (YL1/2)(VMA1864, Abcys, Paris, France), polyclonal anti Glu-α-tubulin antibody(L4) (AbC0101, Abcys, Paris, France), DyLight 488-labeled secondary goatanti-mouse IgG, DyLight 633-labeled secondary goat anti-rabbit IgG (KPL,Gaithersburg, Md., USA), Cy3-conjugated secondary goat anti-Rat IgG,(Jackson ImmunoResearch). 4. Images were collected with a Leica SP5confocal laser scanning microscope (Mannheim, Germany) with 63× oilimmersion optics, laser lines at 488, 561, 633 nm. Identical confocallaser scanning microscopy (CLSM) parameters (e.g., scanning line, laserlight, gain, and offset etc.) were used for control and NAP treatedcells.

Legend: Cortical neurons were treated with vehicle or NAP (10⁻¹⁸M-10⁻⁹M)at DIV 13 for 2 hrs and then fixed and stained for Tyr-tubulin,Glu-tubulin and PSD-95. (a) Representative images of cortical neuronsstained for Tyr-tubulin (red), PSD-95 (green), Glu-tubulin (blue). Bars:10 μm. (b) Comparison of the effect on PSD-95 density. Results show thatthe density of PSD-95 puncta was significantly increased in neuronstreated with NAP for 2 hrs when compared to vehicle controls. Zincreduced cell viability by ˜25 percent (P<0.0001, ANOVA, Dunnett posthoc, n=25 dendrites per treatment). NAP, SKIP and Ac-SKIP (10⁻⁹M) eachseparately significantly increased survival (*P<0.05, ***P<0.001). (c)Comparison of the derivative peptides effect on PSD-95 density. Resultsshow that the density of PSD-95 puncta was significantly increased inneurons treated for 2 hrs with NAPVSIPQ or NAPVSKIPQ when compared tovehicle controls, and was not affected by the NAPVSAIPQ or NAPVAAAAQpeptides. (ANOVA, p<0.0001, n=14-48 dendrites per treatment). (d) 13 DIVcortical neurons were either untreated (UT), treated with transfectionreagent (Lipofectamine RNAiMAX), treated with siRNA against EB3, treatedwith NAP (10⁻¹²M), treated with NAP (10⁻¹²M) and non-targeting controlsiRNA (non), or treated with NAP (10⁻¹²M) and siRNA against EB3. 2 hrstreatment with 10⁻¹²M NAP increased PSD-95 expression by 75% and thiswas completely inhibited by 48 hours EB3 silencing. (ANOVA, p<0.0001,n=18 dendrites per treatment).

FIG. 11: Treatment with NAP increases PSD-95 accumulation in dendriticspines. Methods: 1. Primary cultures of neurons were prepared asfollows: Newborn rats were sacrificed on postnatal day 1. Cerebralcortex tissue was then dissected and dissociated individually from eachpup with the Papain Dissociation System (PDS; Worthington BiochemicalCorporation) according to the manufacturer's instructions (as in FIG.10). 2. Antibodies—monoclonal anti PSD-95 (ab-2723, Abcam) (as in FIG.10), DyLight 488-labeled secondary goat anti-mouse IgG. Hoechst dye wasused to visualize nuclei. 3. Cells were fixed with ice cold methanol,blocked with 2% bovine serum albumin (BSA) and 5% normal goat serum inTris buffered saline containing tween 20 (TBS-T; 20 mM Tris pH 7.5,136.8 mM NaCl, and 0.05% v/v Tween 20), and incubated with primaryantibody followed by the appropriate secondary antibody. 4. Images werecollected with Images were collected with a Leica SP5 confocal laserscanning microscope (Mannheim, Germany) with 40× oil immersion optics,laser lines at 488, 561, 633 nm. Identical confocal laser scanningmicroscopy (CLSM) parameters (e.g., scanning line, laser light, gain,and offset etc.) were used for control and NAP treated cells.

Legend: Representative images showing PSD-95 expression in primarycortical neurons treated with NAP (10⁻¹²M) or vehicle (control).Cortical neurons were treated with vehicle or NAP (10⁻¹²M) at DIV 13 for2 hours and were then fixed and stained for PSD-95. Field view ofprimary cortical neurons (visible light/phase contrast) stained forPSD-95 (green) is seen. This figure differs from FIG. 10, as it isviewed under different magnification and different illumination.

FIG. 12: Proposed mechanism of action for NAP. NAP interacts with EB3 orEB3/EB1 dimer. This interaction promotes and increases other SxIPproteins like p140Cap to interact with EB's. These SxIP proteinsregulate, for example, Src/Fyn which in turn regulates phosphorylationof tau. Alternatively, NAP acts as an interfering peptide that preventsdepolymerizing +TIPs while maintaining MT dynamics.

FIG. 13: Protection of cortical culture by NAP-like peptides.

FIG. 14: NAPVSIPQ affects the microtubule network.

FIG. 15: Rat pheochromocytoma cells (PC 12) were seeded on Poly-D-Lysinecoated 96-well tissue culture dishes in high concentration (30,000cells/well). On the day of the experiment the cells were treated withZnCl₂ (400 μM) either alone or in combination with differentconcentration of SKIP for 4h. After 4h the cell viability was analyzedby MTS assay. (A) The exposure of the PC12 cells to ZnCl₂ induced asignificant reduction in the cell viability. (B) SKIP (SEQ ID NO:6) atconcentrations of 10⁻¹⁵M, 10⁻⁹M showed protection against Znintoxication (significantly higher viability compared to Zn alone). Allvalues are from one experiment performed in hexplicate. The experimentwas repeated several times showing protection at various concentrations.

FIG. 16: SKIP (SEQ ID NO:6) treatment increases the relativediscrimination between novel and familiar objects. Animal performance inthe object recognition memory test is shown. Data are expressed as mean(±SEM) total time (s) spent exploring all objects designated by relativediscrimination index (D1) in Phase 2 (A) and Phase 3 (B). Two identicalobjects were presented during Phase 1, and one of the identical objectswas replaced by a novel object during Phases 2 and 3. The ADNP-deficientmice tended to spend less time in exploring the new objects duringPhases 2 and 3 as compared to control mice (ADNP+/+). SKIP (SEQ ID NO:6)treatment improved short and long term memory for the ADNP-deficientmice. The data was analyzed using the following formula: D1=b−a, when‘a’ designated the time of exploration of the familiar object and ‘b’designated the time of exploration of the novel object. The formulaevaluates the discrimination capacity of the mice between the novelobject and the familiar object. The results were tested statisticallyusing ANOVA test ([*] p<0.05).

FIG. 17: ADNP+/− male mice exhibit spatial learning deficiencies:improvements by SKIP (SEQ ID NO:6) treatment. Two daily water maze testswere performed: first (A) and second (B). Males (ADNP+/+, n=14; ADNP+/−,n=13) were compared. Tests were performed over 5 consecutive days.Latency measured in seconds (mean±S.E.) to reach the hidden platform inits new daily location is depicted. A, ADNP+/− male mice were impairedcompared with control animals. Treatment with SKIP (SEQ ID NO:6) for 1month (twice a day) improved the memory. B, statistically significantdifference (p<0.05, two tailed t-test) between the ADNP+/+ mice andADNP+/− mice on the 5^(th). SKIP (SEQ ID NO:6) treatment resulted inimprovement in the behavior of the ADNP-deficient mice, bringing them tothe control levels.

FIG. 18: ADNP+/− male mice exhibit risky behavior in the elevated plusmaze test. The number of total arm entries (counts) and time spent inthe open-arms for 5 min are presented. Data are expressed as mean±SEM.ANOVA test showed a significant difference (P<0.01) of the time spent inthe open and closed arms between ADNP+/+ mice and ADNP+/− mice. SKIP(SEQ ID NO:6) treatment resulted in improvement in the risky behavior ofthe ADNP-deficient mice.

FIG. 19: Peptide NAPVSGIPQ (SEQ ID NO:5) exhibits protective activity inPC12 cells after exposure to ZnCl₂ (400_(R)M). The same experimentalprocedure was followed as in the experiments shown in FIG. 15. NAPVSGIPQ(SEQ ID NO:5) at concentration of 10⁻¹³M showed protection against Zntoxicity (significantly higher viability compared to Zn alone).

DEFINITIONS

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. For thepurposes of this application, amino acid analogs refers to compoundsthat have the same basic chemical structure as a naturally occurringamino acid, i.e., an a carbon that is bound to a hydrogen, a carboxylgroup, an amino group, and an R group, e.g., homoserine, norleucine,methionine sulfoxide, methionine methyl sulfonium. Such analogs havemodified R groups (e.g., norleucine) or modified peptide backbones, butretain the same basic chemical structure as a naturally occurring aminoacid. For the purposes of this application, amino acid mimetics refersto chemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may include those having non-naturally occurringD-chirality, as disclosed in WO 01/12654, which may improve stability,oral availability and other drug-like characteristics of the compoundcontaining such D-amino acids. In such embodiments, one or more, andpotentially all of the amino acids of the peptides of this inventionwill have D-chirality. The therapeutic use of peptides can be enhancedby using D-amino acids to provide longer half-life and duration ofaction. However, many receptors exhibit a strong preference for L-aminoacids, but examples of D-peptides have been reported that haveequivalent activity to the naturally occurring L-peptides, for example,pore-forming antibiotic peptides, beta amyloid peptide (no change intoxicity), and endogenous ligands for the CXCR4 receptor. In thisregard, NAP and related polypeptides also retain activity in the D-aminoacid form (Brenneman et al., J. Pharmacol. Exp. Ther. 309(3):1190-7(2004); U.S. Pat. No. 8,017,578).

Amino acids may be referred to by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Serine (S), Threonine (T);    -   3) Aspartic acid (D), Glutamic acid (E);    -   4) Asparagine (N), Glutamine (Q);    -   5) Cysteine (C), Methionine (M);    -   6) Arginine (R), Lysine (K), Histidine (H);    -   7) Isoleucine (1), Leucine (L), Valine (V); and    -   8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see, e.g.,        Creighton, Proteins (1984)).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.Generally, a peptide refers to a short polypeptide. The terms apply toamino acid polymers in which one or more amino acid residue is an analogor mimetic of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to an antigen-binding protein having a basic four-polypeptidechain structure consisting of two heavy and two light chains, saidchains being stabilized, for example, by interchain disulfide bonds,which has the ability to specifically bind antigen. Both heavy and lightchains are folded into domains.

The term “antibody” also refers to antigen- and epitope-bindingfragments of antibodies, e.g., Fab fragments, that can be used inimmunological affinity assays. There are a number of well characterizedantibody fragments. Thus, for example, pepsin digests an antibodyC-terminal to the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ can be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially a Fab with part of the hinge region (see, e.g., FundamentalImmunology, Paul, ed., Raven Press, N.Y. (1993), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that fragments can be synthesized de novoeither chemically or by utilizing recombinant DNA methodology. Thus, theterm antibody also includes antibody fragments either produced by themodification of whole antibodies or synthesized using recombinant DNAmethodologies.

The phrase “specifically binds,” when used in the context of describingthe interaction between a protein or peptide and another agent orcompound (e.g., an antibody), refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designated assayconditions, the specified binding agent (e.g., an antibody) binds to aparticular protein at least two times the background and does notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein or a protein but not its similar “sister” proteins. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein or in a particularform. For example, solid-phase ELISA immunoassays are routinely used toselect antibodies specifically immunoreactive with a protein (see, e.g.,Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a descriptionof immunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective bindingreaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The term “subject” or “patient” refers to any mammal, in particularhuman, at any stage of life.

The term “contacting” is used herein interchangeably with the following:combined with, added to, mixed with, passed over, incubated with, flowedover, etc. Moreover, the peptides or other apoptosis modulators of thepresent invention can be “administered” by any conventional method suchas, for example, parenteral (e.g., intravenous, subcutaneous,intradermally or intramuscularly), oral, topical, intravitreal andinhalation (e.g., intranasal) routes.

As used herein “treatment” includes both therapeutic and preventativetreatment of a condition, such as treatment for alleviating ongoingsymptoms and prevention of disease progression or onset of furthersymptoms, or for avoidance or reduction of side-effects or symptoms of adisease. As used herein the term “prevent” and its variations do notrequire 100% elimination of the occurrence of an event; rather, the termand its variation refer to an inhibition or reduction in the likelihoodof such occurrence.

As used herein, “condition” and “disease” include incipient conditionsor disorders, or symptoms of a disease, incipient condition or disorder.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. The term“purified” denotes that protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the protein is at least85% pure, more preferably at least 95% pure, and more preferably atleast 99% pure.

“An amount sufficient (or effective)” or “an effective amount” or a“therapeutically effective amount” is that amount of a therapeutic agentat which the agent exhibits its activity for the intended purpose of itsadministration. In therapeutic applications, an amount adequate toaccomplish this is defined as the “therapeutically effective dose.” Forexample, an effective amount for a neuroprotective agent (e.g., apeptide containing the core amino acid sequence of SEQ ID NO:1) of theinvention is an amount that when administered to a patient sufferingfrom or at risk of developing a disorder involving excessive andunderdesired neuronal cell death (e.g., a neurodegenerative disorder),the agent is capable to reducing or substantially eliminating excessivecell death in neuronal cells, or reducing or substantially eliminatingthe risk of developing a neurodegenerative disorder.

The term “neuroprotective activity” is used in this application to referto a biological activity exhibited by a compound, e.g., a peptide, thatmeasurably reduces, prevents, or eliminates apoptosis in neuronal cellsupon exposure to a toxic agent known to cause death in cells of suchvariety. For example, whether or not a given compound possesses“neuroprotective activity” can be tested and verified by methods knownin the art and/or described herein, including but not limited to, apheochromocytoma (PC 12) cell survival assay involving Zinc toxicity.

As used herein, the term “EB protein” encompasses a group of highlyconserved microtubule plus-end tracking proteins, their homologs,orthologs, and variants. There are three main EB proteins in themammalian species, EB1, EB2, and EB3. The proteins encoded by the MAPREfamily are encompassed within the “EB proteins.” For example, the aminoacid sequences for human and rodent EB proteins and known variants canbe found at the NCBI worldwide website (ncbi.nlm.nih.gov). The GenBankAccession numbers for some rodent EB polynucleotide sequences are setforth in Table 1.

10 human EB protein sequences are available Accession: Q9UPY8.1; GI:20138791 Accession: NP_001243349.1; GI: 374081840 Accession:NP_001137299.1; GI: 219842327 Accession: NP_036458.2; GI: 10800412Accession: Q15555.1; GI: 60390165 Accession: Q15691.3; GI: 20138589Accession: NP_001137298.1; GI: 219842325 Accession: NP_055083.1; GI:10346135 Accession: AAK07557.1; GI: 12751131 Accession: AAK07556.1; GI:12751130 Mouse EB1: Accession: Q61166.3; GI: 60390180 EB2: Accession:Q8R001.1; GI: 60390207 EB3: Accession: Q6PER3.1; GI: 60390186 Rat EB1:Accession: Q66HR2.3; GI: 60389848 EB2: Accession: Q3B8Q0.1; GI:108860788 EB3: Accession: Q5XIT1.1; GI: 60389846

As used in this application, an “increase” or a “decrease” refers to adetectable positive or negative change in quantity from a comparisonbasis, e.g., an established baseline value of the level of an mRNAencoding an EB protein. An increase is a positive change that istypically at least 10%, or at least 20%, or 50%, or 100%, and can be ashigh as at least 2-fold or at least 5-fold or even 10-fold of thecontrol value. Similarly, a decrease is a negative change that istypically at least 10%, or at least 20%, 30%, or 50%, or even as high asat least 80% or 90% of the control value. Other terms indicatingquantitative changes or differences from a comparative basis, such as“more,” “less,” “higher,” and “lower,” are used in this application inthe same fashion as described above. In contrast, the term“substantially the same” or “substantially lack of change” indicateslittle to no change in quantity from the standard control value,typically within ±10% of the standard control, or within ±5%, 2%, oreven less variation from the standard control.

The term “cell death” is used in this application interchangeably withthe term “apoptosis” to refer to a process known as the programmed celldeath (PCD), which involve a series of biochemical events leading tocharacteristic changes in cell morphology and function, and ultimately,to cell death. These changes include blebbing, cell shrinkage, nuclearfragmentation, chromatin condensation, and chromosomal DNAfragmentation. In this application, this term is used in contrast to“necrosis,” which is a form of traumatic cell death that results fromacute cellular injury.

The term “cell plasticity,” as used in this application, refers to acell's ability to alter its functional and/or morphological features inresponse to an internal or external stimulating event. Neuronal orsynaptic plasticity refers to the ability of a neuron cell or synapse tochange its internal characteristic in response to its history. Suchplasticity can include the ability of the entire brain structure and thebrain itself to undergo changes from past experience.

As used herein, the term “expression” encompasses both the transcriptionof a DNA coding sequence into corresponding RNA, indicated by thepresence and quantity of the RNA, and the translation of the encodingRNA into a protein product, indicated by the presence and quantity ofthe protein. In other words, the “expression” of a gene product can bedetermined and quantified at the level of either the corresponding RNAor corresponding protein.

DETAILED DESCRIPTION OF THE INVENTION

Activity-dependent neurotrophic factors (ADNF) are polypeptides thathave neurotrophic or neuroprotective activity as measured with in vitrocortical neuron culture assays described by, e.g., Hill et al., BrainRes. 603:222-233 (1993); Brenneman & Gozes, J Clin. Invest. 97:2299-2307(1996), Gozes et al., Proc. Natl. Acad. Sci. USA 93, 427-432 (1996). Twowell-known ADNF polypeptides comprise an active core site having theamino acid sequence of SALLRSIPA (often referred to as “SAL”) andNAPVSIPQ (often referred to as “NAP”), respectively.

The surprising finding of the present inventors is that theneuroprotective peptides NAP (NAPVSIPQ), NAPVSKIP and SKIP (SEQ ID NO:6)interact with the microtubule (MT) End Binding protein 3 (EB3), whichwill infer interaction with other EB proteins (e.g., EB1 and EB2). Given(1) the structural similarities among different EB proteins, (2) thefact that the SIP motif is required for NAP activity, and (3) that NAPhas a preferential neuroprotection/neurotrophic activity and does notinteract with cancer cells, the inventors hypothesized that NAP(NAPVSIPQ) interacts with EB3 (or with the EB family of proteins). Intheir studies, affinity chromatography showed association of NAP withEB3.

In the studies presented herein, NAP showed significant dose-dependentincrease in PSD-95 expression in dendritic spine like structures incortical neurons in culture. Silencing EB3 mRNA abolished NAP activity,implicating EB3 in the NAP-related neurotrophic effects. Based on NAPstructure, several additional novel peptides derived from hybridsequences of EB3—binding +TIPs and NAP were synthesized including,NAPVSKIPQ; NAPVSAIPQ; NAPVAAAAQ. NAPVSKIPQ mimicked NAP activity,while 1) NAPVSAIPQ, 2) NAPVAAAAQ, 3) NAPVTRIPQ and 4) NAPVSRIPQ wereinactive. NAP has been previously shown to protect against MT breakdownand tubulin aggregation in the presence of toxic concentration of zincthat were associated with neuronal and glial death. In apheochromocytoma (PC12) cell survival assay, the novel NAPVSKIPQprovided protection against zinc toxicity, mimicking NAP activity.Acetyl—NAPVSKIPQ-NH₂ also provided cell protection. Similarly, NAPVSGIPQand the 4-amino acid peptide SKIP (SEQ ID NO:6) provided protection. TheNAP target EB3 (or the EB family of proteins) and interacting peptidesand peptide mimetics are claimed as a discovery platform/assay systemand novel neurotrophic, neuroprotective compounds.

I. Peptides

The peptides of this invention can be obtained by various means wellknown in the art, such as by chemical synthesis or recombinantproduction.

A. Chemical Synthesis

The peptides useful according to this invention can be producedchemically, e.g., by systematically adding one amino acid at a time,followed by screening of the resulting peptide for biological activity,as described herein. In some cases, one or more of the amino acids inthe core active sites may be substituted by a D-amino acid. In addition,various substitutions may be made to amino acid residues outside of thecore sites.

Peptides comprising non-standard amino acids can also be made. In someembodiments, at least one of the amino acids of the active core sequenceis a non-standard amino acid. In some embodiments, 2, 3, 4, 5, or moreof the amino acids is a non-standard amino acid. In some cases, allamino acids are non-standard amino acid (such as D-amino acid) in a coreactive site. Examples of non-standard amino acids arealpha-aminoisobutyric acid, N-methylated amino acids, amino acids with aD chiral center, aza-tryptophan, etc. A wide range of non-standard aminoacids are commercially available, e.g., at Genzyme Pharmaceuticals(Cambridge, Mass.).

Peptide sequences, including those with non-standard amino acids, can begenerated synthetically using commercially available peptidesynthesizers to produce any desired polypeptide (see Merrifield, Am.Chem. Soc. 85:2149-2154 (1963); Stewart & Young, Solid Phase PeptideSynthesis (2nd ed. 1984)). Various automatic synthesizers and sequencersare commercially available and can be used in accordance with knownprotocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis(2nd ed. 1984)). Solid phase synthesis in which the C-terminal aminoacid of the sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids, or non-standard aminoacids, in the sequence is a method for the chemical synthesis of thepeptides of this invention. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, Solid-Phase Peptide Synthesis; pp.3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: SpecialMethods in Peptide Synthesis, Part A.; Merrifield et al 1963; Stewart etal. 1984). The NAP-derived and other peptides of this invention can besynthesized using standard Fmoc protocols (Wellings & Atherton, MethodsEnzymol. 289:44-67 (1997)). Furthermore, liquid phase sequentialsynthesis can be used as well.

B. Recombinant Production

In addition to chemical synthesis, the peptides of this invention,especially those of relatively longer lengths, can be prepared byrecombinant DNA methodology. Generally, this involves creating a nucleicacid sequence that encodes the polypeptide, placing the nucleic acid inan expression cassette under the control of a particular promoter, andexpressing the protein in a host cell. Recombinantly engineered cellsknown to those of skill in the art include, but are not limited to,bacteria, yeast, plant, filamentous fungi, insect (especially employingbaculoviral vectors), and mammalian cells.

The recombinant nucleic acids are operably linked to appropriate controlsequences for expression in the selected host. For E. coli, exemplarycontrol sequences include the T7, trp, or lambda promoters, a ribosomebinding site and, optionally, a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and,optionally, an enhancer, e.g., derived from immunoglobulin genes, SV40,cytomegalovirus, etc., a polyadenylation sequence, and splice donor andacceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by methods such as, for example, the calcium chloridetransformation method for E. coli and the calcium phosphate treatment orelectroporation methods for mammalian cells. Cells transformed by theplasmids can be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo, and hyg genes.

Once expressed, the recombinant polypeptides can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, e.g., Scopes, PolypeptidePurification (1982); Deutscher, Methods in Enzymology Vol. 182: Guide toPolypeptide Purification (1990)). Optional additional steps includeisolating the expressed polypeptide to a higher degree, and, ifrequired, cleaving or otherwise modifying the peptide, includingoptionally renaturing the polypeptide.

One of skill can select a desired polypeptide of the invention basedupon the sequences provided and upon knowledge in the art regardingproteins generally. Knowledge regarding the nature of proteins andnucleic acids allows one of skill to select appropriate sequences withactivity similar or equivalent to the polypeptides disclosed herein.

One of skill will recognize many ways of generating alterations in anucleic acid sequence encoding a given peptide sequence. Polypeptidesequences can also be altered by changing the corresponding nucleic acidsequence and expressing the polypeptide. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other known techniques (see Giliman &Smith, Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)).

After chemical synthesis, recombinant expression or purification, thepolypeptide(s) may possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it is helpful to denature and reduce the polypeptide and then to causethe polypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing peptides and inducing re-folding are known tothose of skill in the art (see Debinski et al., J. Biol. Chem.268:14065-14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:581-585(1993); and Buchner et al., Anal. Biochem. 205:263-270 (1992)). Debinskiet al., for example, describe the denaturation and reduction ofinclusion body peptides in guanidine-DTE. The peptide is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

The peptides described in this invention can be evaluated by screeningtechniques in suitable assays for the desired characteristic, e.g.,promoting cell survival/plasticity or inhibiting/reducing cell deathupon external assault. For instance, the peptides, as well as othercompounds that modulate cell death/survival, described in the presentinvention can be screened by employing suitable assays described hereinor known to those skilled in the art.

One of skill will recognize that modifications can be made to thepolypeptides without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orintake of the polypeptide by the target cells or tissue. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

C. Modification of Peptides

In some cases it might be desirable to further modify the peptides ofthe present invention, for example, to increase the stability orbioavailability of the peptide. One example of such modification isacetylation of the peptides at a suitable site (e.g., the N-terminus ofthe peptide or the internal NH₂ group on a Lys residue). Acetylation canbe achieved by chemical reaction or enzymatic reaction, both methodsknown in the art.

Other examples of peptide modification include glycosylation,PEGylation, lipidation, amidation, or addition of a tag sequence forease of purification and subsequent handling. For instance, a peptidemay be amidated at its C-terminus to form —CO—NH₂ (replacing the OH),especially in multiple natural peptides. Furthermore, it is possible toplace an additional peptide tail to a peptide of this invention toimprove certain desired characteristics, e.g., to increase permeabilityof the peptide.

D. Functional Assays of Peptides and Other Compounds with Anti-ApoptosisActivity

The novel peptides and other compounds described herein are useful forthe method of this invention due to their activity in promoting cellsurvival, or suppressing cell death upon exposure to cytotoxicity.Furthermore, the present invention allows for initial screening ofadditional compounds, which can be widely diverse in their chemicalnature, as possible modulators of cell survival and cell death.Functional assays are performed to verify the biological activity ofthese peptides or other possible modulators.

A variety of cell culture-based methods are known in the art, as well asdescribed in this application, for testing and demonstrating acompound's potential effects on cell survival. For instance, a cellviability assay using a suitable cell type (e.g., a cultured neuronalcell line such as PC12 cells) may be employed to compare the cell count,the rate of cell division, and/or the level of cell metabolism in thepresence or absence of a potential modulator under test conditions(e.g., when cells are subject to treatment of a toxic agent such as Zincat a harmful concentration). While a potential modulator of cellsurvival could have either positive or negative effects on the cells'susceptibility to cell death, when the presence of a compound leads toincreased cell survival, the compound is deemed an inhibitor orsuppressor of cell death; conversely, when the presence of a compoundleads to decreased cell survival, the compound is deemed an enhancer ofcell death.

E. Functional Assays for Compounds Promoting Cell Plasticity

The novel peptides and other compounds described herein are also usefulfor the method of this invention due to their activity in promoting,protecting, or increasing cell plasticity, especially neuronalplasticity or synaptic plasiticy. Furthermore, the present inventionallows for initial screening of additional compounds, which can bewidely diverse in their chemical nature, as possible modulators of cellplasticity. Functional assays are performed to verify the biologicalactivity of these peptides or other possible modulators.

A variety of cell culture-based methods are known in the art, as well asdescribed in this application, for testing and demonstrating acompound's potential effects on cell plasticity. For instance, a PSD-95expression assay using primary cortical neuron cultures established fromsuitable animals may be performed to confirm a compound's activity inpromoting cell plasticity. More specifically, primary cultures ofneurons are prepared by first taking cerebral cortex tissue from newbornrats on postnatal day 1. The cerebral cortex tissue is then dissectedand dissociated individually from each animal. Cells are fixed with icecold methanol, blocked (e.g., with bovine serum albumin (BSA) and normalgoat serum), and incubated with primary antibody (e.g., monoclonalantibody against PSD-95), optionally followed by the appropriatesecondary antibody for imaging purposes. Images of primary neuronalcultures are taken and compared between cultures with or without beingexposed to a test compound. An increased PSD-95 expression is indicativeof the test compound being capable of promoting or protecting cellplasticity, especially neuronal plasticity.

II. Screening Methods for Identifying Modulators of Cell Survival orPlasticity

A. Screening Methods Based on Binding with EB Proteins

By illustrating the role of EB proteins during the cell death/survivalor plasticity process, the present invention allows identification ofmodulators of cell susceptibility to apoptosis or cell plasticity byscreening among candidate compounds for such potential modulators basedon such compounds' physical interaction or binding with at least one ofthe EB proteins, e.g., a human or rodent version of an EB protein.

Various methods are known in the art for detecting binding between aknown protein and potential “binders” of any chemical nature. Suchmethods are also described in detail in this application. For example, acandidate compound may be immobilized on a solid substrate, and asolution containing a suitable EB protein is incubated with thesubstrate under conditions that permit the binding between the EBprotein and a potential “binder” molecule. After a proper washing step,the presence of the EB protein is then detected, e.g., by an antibodythat specifically recognizes the EB protein, or by the presence of adetectable label previously conjugated to the EB protein. The binding ofthe EB protein and any given test compound would indicate the compoundto be a potential modulator of cell death/cell survival. One example ofsuch binding assay is the affinity chromatography described in theexamples of this application. Chips containing a large, immobilized testcompound library, e.g., protein arrays or proteomic chips, will beuseful for this purpose. Typically, a positive control, such as apeptide known to bind to an EB protein (e.g., NAP peptide that is knownto bind EB3 protein), as well as a negative control, such as a peptideknown to not bind to EB proteins, are included in the screen assay toensure accurate determination of binding between a candidate agent andan EB protein.

B. Screening Methods Based on Effects on Expression of EB Proteins

The present invention further provides for the screening of potentialmodulators of cell death/survival or cell plasticity from a largecollection of molecules of any chemical nature based on a modulator'seffects on the expression of an EB protein. Any such effect on EBprotein expression may be detected and monitored at either mRNA level orprotein level.

Similar to the EB protein binding assay format, candidate compounds maybe screened as a first, preliminary step to quickly identify aspotential modulators of cell death/survival or cell plasticity, whichmay then be subject to further testing for functional verification. Insome cases, the effect on EB protein expression is tested in acell-based assay system, where a suitable cell type (e.g., a neuronalcell) that endogenously expresses one or more EB proteins is exposed toa candidate compound under conditions that permits EB protein expressionin the cells. The level of the EB protein and/or mRNA is then measuredand compared between cell samples where the candidate compound ispresent or absent. When an increased expression of EB protein or mRNA isdetected, the test compound is deemed a potential modulator thatpromotes cell survival or cell plasticity, or an inhibitor or suppressorof cell death. Conversely, if a decreased expression of EB protein ormRNA is detected, the test compound is deemed a potential modulator thatincreases cell susceptibility to cell death or inhibits cell plasticity,i.e., a promoter of cell death or inhibitor of cell plasticity.

Also similar to the EB protein binding assays described above,appropriate positive and negative controls are often used in the assaysto ensure the proper operation of the experimental system. Finally, apreliminarily identified cell death/survival or plasticity modulatorbased on its effect on the expression of an EB protein is subject tofunctional verification using the functional assays described in thelast section.

C. Functional Assays

The screening assays described above often serve as a useful tool topreliminarily identify, from a large pool of candidate compounds,possible modulators of cell survival or cell plasticity. To fully verifyand more precisely determine the functional effects of these potentialmodulators, functional assays described in the last section aretypically performed subsequent to the initial screening step.

III. Pharmaceutical Compositions and Administration

The pharmaceutical compositions comprising the modulators that promotecell survival or modulate cell plasticity as described in thisapplication (e.g., a peptide having the amino acid sequence set forth inSEQ ID NO:2 or SEQ ID NO:5 or the 4-amino acid peptide SKIP, includingthe version with N-terminus acetylation or lipophilization and/orC-terminus amidation, as well as all D-amino acids, or any one of thecompounds named in Table 4) are suitable for use in a variety of drugdelivery systems. The polypeptides can be administered systemically,e.g., by injection (intravenous, subcutaneous, intradermal, orintramuscular), or by oral administration, or by nasal administration,or a local administration such as using a dermal patch, under the tonguepellet etc. The methods for various routes of delivery are well known tothose of skill in the art.

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (17th ed. 1985)). For a brief reviewof methods for drug delivery, see Langer, Science 249:1527-1533 (1990).Suitable dose ranges are described in the examples provided herein, aswell as in WO96/11948.

As such, the present invention provides for therapeutic compositions ormedicaments comprising one or more of the polypeptides describedhereinabove in combination with a pharmaceutically or physiologicallyacceptable excipient, wherein the amount of polypeptide is sufficient toprovide a therapeutic effect, e.g., to improve the neurodegenerativecondition a patient is receiving the treatment for.

In a therapeutic application, the modulators of the present inventionare embodied in pharmaceutical compositions intended for administrationby any effective means, including parenteral, topical, oral, nasal,pulmonary (e.g. by inhalation) or local administration. Nasalpumps/sprays, eye drops, and topical patches can be used.

The invention provides compositions for parenteral administration thatcomprise a solution of a modulator (e.g., a peptide comprising SKIP), asdescribed above, dissolved or suspended in an acceptable carrier, suchas an aqueous carrier. Parenteral administration can comprise, e.g.,intravenous, subcutaneous, intradermal, intramuscular, or intranasaladministration. A variety of aqueous carriers may be used including, forexample, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronicacid and the like. These compositions may be sterilized by conventional,well known sterilization techniques or, they may be sterile filtered.The resulting aqueous solutions may be packaged for use as is orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions including pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, such as, for example, sodium acetate, sodium lactate, sodiumchloride potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be usedthat include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the modulators are preferably supplied infinely divided form along with a surfactant and propellant. Accordingly,in some embodiments, the pharmaceutical composition comprises asurfactant such as a lipophilic moiety to improve penetration oractivity. Lipophilic moieties are known in the art and described, e.g.,in U.S. Pat. No. 5,998,368. The surfactant must be nontoxic, andpreferably soluble in the propellant. Representative of such agents arethe esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. A carrier can also be included,as desired, as with, e.g., lecithin for intranasal delivery. An exampleincludes a solution in which each milliliter included 7.5 mg NaCl, 1.7mg citric acid monohydrate, 3 mg disodium phosphate dihydrate and 0.2 mgbenzalkonium chloride solution (50%) (see, e.g., Gozes et al., J MolNeurosci. 19:167-70 (2002)).

In therapeutic applications, the modulators of the invention areadministered to a patient in an amount sufficient to reduce or eliminatesymptoms of a condition involving undesirable cell death, such as aneurodegenerative disorder. An amount adequate to accomplish this isdefined as “therapeutically effective dose.” Amounts effective for thisuse will depend on, for example, the particular peptide employed, theparticular form of a pharmaceutical composition in which the peptide ispresent (e.g., a peptide of SEQ ID NO:2 or SKIP v. an acetylated peptideof SEQ ID NO:2 or SKIP), the type of disease or disorder to be treatedand its severity, the manner of administration, the weight and generalstate of health of the patient, and the judgment of the prescribingphysician.

For example, an amount of a cell survival or cell plasticity modulator,e.g., a peptide of SEQ ID NO:2 or SKIP or any one of the compoundslisted in Table 4, falling within the range of a 100 ng to 30 mg dosegiven intranasally once a day would be a therapeutically effectiveamount. Alternatively, dosages may be outside of this range when on adifferent schedule (such as by injection or oral ingestion). Forexample, dosages can range from 0.0001 mg/kg to 10,000 mg/kg, and can beabout 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 50 mg/kg or 500 mg/kgper dose. Doses may be administered hourly, every 4, 6 or 12 hours, withmeals, daily, every 2, 3, 4, 5, 6, or 7 days, weekly, every 2, 3, 4weeks, monthly or every 2, 3 or 4 months, or any combination thereof.The duration of dosing may be single (acute) dosing, or over the courseof days, weeks, months, or years, depending on the condition to betreated. Those skilled in the art can determine the suitable dosage andadministration frequency depending on the particular circumstances ofindividual patients.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 Microtubule End Binding (EB) Proteins—theNeuroprotective/Neurotrophic NAP (Davunetide) Target Introduction

The plus ends of growing MTs accumulate a diverse group of MT-associatedproteins, collectively referred to as the plus-end-tracking proteins(+TIPs). +TIPs encompass a large number of unrelated proteins, motor andnonmotor proteins, regulatory proteins, and adaptor proteins. One +TIPbinding protein family is the end-binding (EB) protein family. Likeother +TIPs, EB proteins mediate interactions between the ends of MTs,organelles, and protein complexes as well as altering MT stability. EB3is preferentially expressed in the CNS and is used to track MT dynamics(E. S. Sweet et al., J Neurosci 31, 1038 (Jan. 19, 2011)). EB3 furtherinteracts with post synaptic density protein, PSD-95 at the level of thedendritic modeling and plasticity (E. S. Sweet et al., J Neurosci 31,1038 (Jan. 19, 2011)). Thus, EB3 plus-end decorate MTs, control actindynamics and regulate spine morphology and synaptic plasticity, throughinteraction with PSD-95, and NMDA receptor activation (L. C. Kapitein etal., J Neurosci 31, 8194 (Jun. 1, 2011)). EB1 was shown to interact witha conserved binding site in +TIPS—namely, SxIP (S. Honnappa et al., Cell138, 366 (Jul. 23, 2009)). Similarly, it was recently shown that plexinsmembrane proteins associated with neurite growth interact with the threeEB proteins with a SxIP (SGIP) motif (P. Laht, K. Pill, E. Haller, A.Veske, Biochim Biophys Acta, (Feb. 21, 2012)). Unlike EB1, EB3 showsspecificity to the nervous system. On the other hand, it has beenreported that EB3 and EB1 heterodymerize to form a dimer, and EBproteins exhibit intricate control on each other (e.g.,www.ncbi.nlm.nih.gov/pubmed/19255245). As such, EB1 may be required forthe biological activity of EB1/EB3 dimer.

The EB protein family is an evolutionarily conserved family of proteins,which in mammals are encoded by the MAPRE gene family and include EB1,EB2 (RPI) and EB3 (J. P. Juwana et al., Int J Cancer 81, 275 (Apr. 12,1999); L. K. Su, Y. Qi, Genomics 71, 142 (Jan. 15, 2001)). In animalcells, EB proteins may constitute the “core” of the plus end complexbecause they interact directly with most other known TIPs includingdynactin large subunit p150Glued, APC, CLASPs, spectraplakins, RhoGEF2,and a catastrophe-inducing kinesin KLP10A (J. M. Askham, K. T. Vaughan,H. V. Goodson, E. E. Morrison, Mol Biol Cell 13, 3627 (October, 2002);W. Bu, L. K. Su, The Journal of biological chemistry 278, 49721 (Dec.12, 2003); L. A. Ligon, S. S. Shelly, M. Tokito, E. L. Holzbaur, MolBiol Cell 14, 1405 (April, 2003); S. L. Rogers, U. Wiedemann, U. Hacker,C. Turck, R. D. Vale, Curr Biol 14, 1827 (Oct. 26, 2004); S. Honnappa,C. M. John, D. Kostrewa, F. K. Winkler, M. 0. Steinmetz, EMBO J 24, 261(Jan. 26, 2005); V. Mennella et al., Nat Cell Biol 7, 235 (March, 2005);Y. Mimori-Kiyosue et al., The Journal of cell biology 168, 141 (Jan. 3,2005); K. C. Slep et al., The Journal of cell biology 168, 587 (Feb. 14,2005)). EB1 was initially identified in a yeast two hybrid screen as aprotein that interacts with the C-terminus of the adenomatous polyposiscoli (APC) tumor suppressor protein, EB1 is also a specific marker ofgrowing MT tips (L. K. Su et al., Cancer research 55, 2972 (Jul. 15,1995)). RNA interference mediated deletion of EB1 from cells leads to anincrease time that a MT spends pausing and EB1 also increases rescuefrequency as well as reduces catastrophe frequency and depolymerizationrates. EB1 protein links MT to actin protrusion and cell polaritythrough signaling pathways involving PKC (J. M. Schober, G. Kwon, D.Jayne, J. M. Cain, Biochemical and biophysical research communications417, 67 (Jan. 6, 2012)). Overall, EB1 recently emerged as a masterregulator of dynamic +TIP interaction networks at growing MT ends (S.Honnappa, C. M. John, D. Kostrewa, F. K. Winkler, M. O. Steinmetz, EMBOJ 24, 261 (Jan. 26, 2005); N. Galjart, F. Perez, Curr Opin Cell Biol 15,48 (February, 2003); S. Honnappa et al., Mol Cell 23, 663 (Sep. 1,2006)). The EB proteins are often small globular dimers that contain twohighly conserved domains that are connected by a linker sequence. TheN-terminal part of the EB protein consists of a calponin homology (CH)domain, which is necessary and sufficient for binding to MTs andrecognizing growing MT ends (Y. Komarova et al., The Journal of cellbiology 184, 691 (Mar. 9, 2009)). The second domain is a coiled coilregion, which determines their dimerization (S. Honnappa, C. M. John, D.Kostrewa, F. K. Winkler, M. O. Steinmetz, EMBO J 24, 261 (Jan. 26,2005); K. C. Slep et al., The Journal of cell biology 168, 587 (Feb. 14,2005)). EB3 is a homologue of EB1 and shares a 54% identity to EB1. EB3is especially abundant in the central nervous system, where it binds toAPC2/APCL, the brain specific form of APC, and it is expressed to lesserdegree in muscles (A. Straube, A. Merdes, Curr Biol 17, 1318 (Aug. 7,2007); H. Nakagawa et al., Oncogene 19, 210 (Jan. 13, 2000)).EB3-depleted cells show disorganized MT and fail to stabilize polarizedmembrane protrusions. EB3 is necessary for the regulation of MT dynamicsand MT capture at the cell cortex (A. Straube, A. Merdes, Curr Biol 17,1318 (Aug. 7, 2007)). Drebrin, an F-actin-associated protein, bindsdirectly to EB3. In growth cones, this interaction occurs specificallywhen drebrin is located on F-actin in the proximal region of filopodiaand when EB3 is located at the tips of MT invading filopodia. When thisinteraction is disrupted, the formation of growth cones and theextension of neurites are impaired. Drebrin targets EB3 to coordinateF-acting MT interactions that underlie neuritogenesis (S. Geraldo, U. K.Khanzada, M. Parsons, J. K. Chilton, P. R. Gordon-Weeks, Nat Cell Biol10, 1181 (October, 2008)). EB3 is a major regulator of spine plasticityby influencing actin dynamics within the dendritic spine. EB3 bindsdirectly to p140Cap, which is localized at the postsynaptic density andis an inhibitor of Src tyrosine kinases (J. Jaworski et al., Neuron 61,85 (Jan. 15, 2009)). All mammalian EB proteins are very similar instructure, but it appears that EB2 is the most divergent family membershowing differences in expression, as well as interactions with MTs andbinding partners (E. E. Morrison, Cell Mol Life Sci 64, 307 (February,2007)). Protein depletion and rescue experiments showed that EB1 andEB3, but not EB2, promote persistent MT growth by suppressingcatastrophes (Y. Komarova et al., Mol Biol Cell 16, 5334 (November,2005)). Where investigated, it appears that interactions identified forEB1 can be replicated by EB3 but not EB2. It was shown that CLIP-170 anda closely related protein CLIP-115 bind directly to EB1 and EB3 whiledisplaying a lower affinity for EB2. This interaction depends on the Cterminal tails of the EB proteins, which are strikingly similar to thoseof alpha tubulin (Y. Komarova et al., Mol Biol Cell 16, 5334 (November,2005)). EB1 and EB3 increase the number of neurites in PC12 cells whileEB2 blocks neurite elongation (V. Laketa, J. C. Simpson, S. Bechtel, S.Wiemann, R. Pepperkok, Mol Biol Cell 18, 242 (January, 2007)). EB1preferentially heterodimerizes with EB3, while, EB2 does not seem toform heterotypic complexes. Heterotypic complex formation between EB1and EB3, thus, generates an additional EB variant which is expected todisplay yet a different functional profile when compared with itshomotypic counterparts. These findings also suggest that EBs are notpresent in separate pools but, rather, form a common pool undergoingcontinuous exchange within the cytoplasm. A consequence of thisconsideration is that in cells that co-express different EB species,their functions cannot be contemplated and analyzed separately from eachother (C. O. De Groot et al., The Journal of biological chemistry 285,5802 (Feb. 19, 2010)).

EB proteins bind to multiple partners and until now two major bindingmodes have been described: through the association of a linear SxIPmotif with the hydrophobic cavity of the EBs and through the binding ofCAP-Gly domains to the C-terminal EEY motif of the EBs. Using live cellimaging experiments and in vitro reconstitution assays, it wasdemonstrated that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), isused by numerous +TIPs, including APC, CLASPs (CLIP associatingproteins), MCAK (mitotic centromere associated kinesin), TIP150, MACF(MT-actin crosslinking factor), STIM1 (stromal interaction molecule 1),p140Cap, Navigators, melanophilin, RhoGEF2, CDK5RAP2 and DDA3, forlocalization to MT tips in an EB1-dependent manner. Highly conservedC-terminal domain of EB1 recognizes this short linear sequence motiffound in a large number of important +TIPs for MT plus-end tracking. Themost prominent contacts involve Ser5477, Ile5479, and Pro5480, whichoccupy the positions 1, 3, and 4 of the SxIP motif (S. Honnappa et al.,Cell 138, 366 (Jul. 23, 2009)). A recent report showed that the serineresidues around the SxIP motifs of CLASP2 are phosphorylated by GSK3f3,disrupting MT plus end tracking (P. Kumar et al., The Journal of cellbiology 184, 895 (Mar. 23, 2009)). These data support the view thatphosphorylation in the vicinity of SxIP motifs negatively regulates thelocalization of +TIPs to MT ends by decreasing their affinity to EB1 (S.Honnappa et al., Cell 138, 366 (Jul. 23, 2009)).

Given the: (1) structural similarities between the different EB proteinsand the lately found plexin binding to EBs, (2) the fact that the SIPmotif is required for NAP activity(M. F. Wilkemeyer et al., Proc NatlAcad Sci USA 100, 8543 (Jul. 8, 2003)), (3) the fact that the SIP motifis also found in activity-dependent neuroprotective factor(ADNF)—SALLRSIPA (M. Bassan et al., J Neurochem 72, 1283 (March, 1999)),(P. Laht, K. Pill, E. Haller, A. Veske, Biochim Biophys Acta, (Feb. 21,2012)) that NAP (as well as SALLRSIPA) have a preferentialneuroprotection/neurotrophic activity (I. Gozes et al., J Mol Neurosci20, 315 (2003); I. Gozes, I. Spivak-Pohis, Curr Alzheimer Res 3, 197(July, 2006)), and interact with MTs (M. Holtser-Cochav, I. Divinski, I.Gozes, J Mol Neurosci 28, 303 (2006)), the present inventorshypothesized that NAP (NAPVSIPQ) interacts with EB3. Given the fact thatEB3 can form dimers with EB1, and interaction with EB1 is alsoenvisaged. EB2 blocks neurite elongation (V. Laketa, J. C. Simpson, S.Bechtel, S. Wiemann, R. Pepperkok, Mol Biol Cell 18, 242 (January,2007)) and hence—if associated with NAP activity, the manner ofinteraction is different.

As eluded to above, NAP has been previously shown to protect against MTbreakdown and tubulin aggregation in the presence of toxic concentrationof zinc that were associated with neuronal and glial death(I. Divinski,M. Holtser-Cochav, I. Vulih-Schultzman, R. A. Steingart, I. Gozes, JNeurochem 98, 973 (August, 2006); I. Divinski, L. Mittelman, I. Gozes, JBiol Chem 279, 28531 (Jul. 2, 2004)).

Materials and Methods

Bioinformatics—pattern search:http://www.genome.jp/tools/motif/http://wwvv.algosome.com/resources/human-proteome/motif-pattern-matcher.htmlhttp://pir.georgetown.edu/pirwww/search/pattern.shtml#http://prosite.expasy.org/cgi-bin/prosite/PSScan.cgihttp://bioware.ucd.ie/˜compass/cgi-bin/formParser.py

Cell Culture Systems P19 Cells

Mouse embryonic teratocarcinoma cells (P19 cells) were obtained from theAmerican Type Culture Collection (ATCC, Bethesda, Md., USA); an initialcontrol batch was a kind gift of Dr. Roi Atlas and the late Prof. IrithGinzburg from the Weizmann Institute of Science, Rehovot, Israel. P19cells were grown in minimal essential medium (alpha-MEM, BiologicalIndustries, Beit Haemek, Israel) containing 5% fetal calf serum, 100U/ml penicillin and 0.1 mg/ml streptomycin (Biological Industries) in a5% CO₂ incubator at 37° C.

Neuronal or Cardiomyocyte Induced P19 Differentiation

For induction of differentiation, lx10⁶ P19 cells were cultured in 90 mmbacteriological grade dishes with their usual growth medium andsupplemented with 1 μM all-trans retinoic acid (RA, Sigma, St. Louis,Mo., USA) to induce neuronal and astroglial differentiation (E. M.Jones-Villeneuve, M. W. McBurney, K. A. Rogers, V. I. Kalnins, J CellBiol 94, 253 (August, 1982)) or 0.8% dimethylsulfoxide (DMSO, Sigma) toinduce cardiac and skeletal muscle differentiation, as previouslydescribed (A. Habara-Ohkubo, Cell Struct Funct 21, 101 (April, 1996); I.S. Skerjanc, Trends Cardiovasc Med 9, 139 (July, 1999); M. A. van derHeyden, L. H. Defize, Cardiovasc Res 58, 292 (May 1, 2003); Mandel etal., J. Mol. Neurosci. 35:127, 2008). Four days later, cell aggregateswere suspended with trypsin-C (Biological Industries) and transferred topoly-L-lysine (Sigma) coated tissue culture dishes. The cells were grownin RA/DMSO-free Dulbecco's modified Eagle's medium (DMEM) containing2.5% fetal calf serum, 4 mM L-glutamine and antibiotics (BiologicalIndustries) for additional four days to induce neuronal and astroglialor cardiac and skeletal muscle phenotype. As controls to thedifferentiated conditions, both non-differentiated cells as well ascells that went through the differentiation process in the absence ofthe inducer were evaluated.

Rat Cerebral Cortical Astrocyte Cell Cultures

Newborn rats (Harlan, Jerusalem, Israel) were sacrificed bydecapitation, and the brain was removed (D. E. Brenneman et al., JPharmacol Exp Ther 309, 1190 (June, 2004)). The tissue was minced withscissors and placed in Hank's balanced salts solution 1 (S1), containingHBSS (Biological Industries, Beit Haemek, Israel), 15 mM HEPES buffer,pH 7.3 (Biological Industries, Beit Haemek, Israel), and 0.25% trypsin(Biological Industries) in an incubator at 37° C. 5% CO₂ for 20 min. Thecells were then placed in 5 mL of solution 2 (S2) containing 10% heatinactivated fetal serum (Biological Industries), 0.1% gentamycinsulphate solution (Biological Industries), and 0.1%penicillin-streptomycin-nystatin solution (Biological Industries) inDulbecco's modified Eagle's medium (DMEM, Sigma, Rehovot, Israel). Thecells were allowed to settle and are then transferred to a new tubecontaining 2.5 mL of S2 and triturated using a Pasteur pipette. Celldensity was determined using a hemocytometer (Neubauer Improved,Germany) and 15×10⁶ cells/15 mL S2 are inoculated into each 75-cm² flask(Corning, Corning, N.Y., USA). Cells are incubated at 37° C. 10% CO₂.The medium was changed after 24 h, and cells are grown until confluent.

Rat Cerebral Cortical Astrocyte Cell Subcultures

The flasks were shaken to dislodge residual neurons and oligodendrocytesthat may be present. Flasks were then washed with 10 mL cold HBSSx1,HEPES 15 mM. 5 mL of versene-trypsin solution (BioLab, Jerusalem,Israel) were added to each flask, and the flasks are incubated at roomtemperature for 5 min to remove astrocytes. The flasks were shaken todislodge the cells. The versenetrypsin solution was neutralized with 5mL of S2. The cell suspension was collected and centrifuged at 100 g for10 min. The supernatant was removed and the cells resuspended in S2.Cells were inoculated into 75-cm² flasks or plated in 35-mm dishes(Corning, Corning, N.Y., USA) and incubated until confluence at 37° C.10% CO₂.

Primary Neuronal Cultures

Cerebral cortex tissue from newborn rats was dissected and dissociatedindividually from each pup with the Papain Dissociation System (PDS),(Worthington Biochemical Corporation) according to the manufacturer'sinstructions. Cortical neurons were maintained in Neurobasal medium(NB), (Gibco) supplemented with NeuroCult B27-SM1 (STEMCELL), 1%Glutamax (Gibco), and grown on poly-D-lysine-coated cell culture glasscover slips. The cells were incubated in 5% CO₂ in a humidifiedincubator at 37° C.

PC12 Cells

PC12 cells (Pheochromocytoma cells) (ATCC, Bethesda, Md., USA) wereseeded at 3×104 cells/cm² on poly-L-Lysine coated plastic tissue culturedishes (Corning, Lowell, Mass., USA) to form an adherent monolayer.Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% horse serum (HS), 5% fetal calf serum (FCS), 2 mMglutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (BiologicalIndustries, Beit Haemek, Israel). PC12 differentiation was induced bynerve growth factor (NGF, Sigma) at concentrations of 50 ng/ml byreplacing half of the medium every other day until the cells acquired adifferentiated morphology. Differentiated cells were defined as bearingtwo or more neurites with lengths equal to or more than twice thediameter of the cell body (P. Lamoureux et al., The Journal of cellbiology 110, 71 (January, 1990)). The cells were incubated in 5% CO₂ ina humidified incubator at 37° C. The cells were sub-cultured every 5days at a 4:1 split ratio by pipetting. The medium was changed every 2or 3 days after adhesion.

NIH3T3 Cells

NIH3T3 (mouse fibroblasts) (ATCC, Bethesda, Md., USA) were cultured inDMEM containing 10% heat inactivated fetal calf serum, 2 mM L-Glutamine,100 units/mL penicillin, and 0.1 mg/mL streptomycin (BiologicalIndustries) in 5% CO₂ at 37° C. (growth conditions). Every 3-4 dayscells were split using trypsin-EDTA solution B (Biological Industries).

Immunostaining

Cultured cells plated on glass coverslips were fixed and permeabilizedsimultaneously, with 3% paraformaldehyde, 0.075% glutaraldehyde (FlukaBiochemika) in MT-buffer (80 mM PIPES pH 6.8, 1 mM MgCl2, 2 mM EGTA, 5%Glycerol) with 0.5% TritonX-100, for 10 min, quenched with 1 mg/ml NaBH4in PBS, blocked with 2% BSA and 5% normal goat serum in TBS-T (20 mMTris pH 7.5, 136.8 mM NaCl, and 0.05% v/v Tween 20), and incubated withprimary antibodies followed by the appropriate secondary antibodies.Nuclei were visualized with Hoechst dye.

Confocal Microscopy and Image Analysis

Images were collected with a Leica SP5 confocal laser scanningmicroscope (Mannheim, Germany) with 63× oil immersion optics, laserlines at 488, 561, 633 nm or with LSM 510 META (Zeiss, Jena, Germany)confocal laser scanning microscope with 63× oil immersion optics, laserlines at 488, 568, 633 nm. When comparing fluorescence intensities,identical CLSM parameters (e.g., scanning line, laser light, gain, andoffset etc.) were used. All of the fluorescent signals acquired wereabove the autofluorescent background as measured from a control slidestained with secondary antibody without a primary antibody. To comparethe relative levels of protein expression, the average integrateddensity (AID) image analysis procedure was used for cell immunostains.In brief, integrated density is defined by the sum of the values of thepixels in the selected region of interest (ROI). This is equivalent tothe product of Area and Mean Gray Value. AID for the positive stainedarea was determined by measuring the fluorescent intensity of the ROI,which is above the positive cut-off intensity. Positive cut-offintensities were determined based on the fluorescence intensitieshistogram for each antibody staining. For measurements of the MTcontaining area in a given cell, the chosen focal plane was the oneshowing the maximal area on the z-axis (focal axis). Analysis was doneusing the MICA software (Cytoview, Petach Tikva, Israel) and ImageJ(NIH, Bethesda, Md., USA).

Peptides

Peptides were purchased form Hy-Labs (Rehovot Israel). All peptides weredissolved in double distilled water to a concentration of 1 mM and thendiluted in water in 1:10 steps up to the required concentration.

List of Peptides:

Short name Sequence NAP/SIP NAPVSIPQ SKIP NAPVSKIPQ SAIP NAPVSAIPQ AAAANAPVAAAAQ SRIP NAPVSRIPQ TRIP NAPVTRIPQ Ac-SKIP Acetyl-NAPVSKIPQ-NH₂SKIP1 SKIP SGIP SGIP NAPVSGIP NAPVSGIP

Antibodies and Other Cell Staining Molecules Primary Antibody List:

Monoclonal anti-βIII-tubulin antibody (T8578, Sigma, Rehovot, Israel),monoclonal anti Tyr-α-tubulin antibody (YL1/2) (VMA1864, Abcys, Paris,France), polyclonal anti Glu-α-tubulin antibody (L4) (AbC0101, Abcys,Paris, France), monoclonal anti-tau (tau-5) (ab80579, Abcam, Mass.,USA), monoclonal anti-tau (tau-5) (Biosource International, Camarillo,Calif., USA), monoclonal anti-total tau (AT-5004, MBL, Billerica, Mass.,USA), monoclonal [TAU-5] anti tau (ab80579, Abcam), polyclonal antiMAPRE3 (ab13859, ab99287, Abcam), monoclonal anti PSD-95 (ab-2723,Abcam), anti actin (ab1801, Abcam, Mass., USA), monoclonal antia-tubulin (DM1A) (T6199, Sigma, Rehovot, Israel).

Other Tracers:

Sulfonated DiI: DiIC18(5)-DS/5 mg (D12730, Invitrogen, NY, USA), alipophilic tracer: long-chain dialkylcarbocyanines, DiI, is used asanterograde and retrograde neuronal tracer in living (M. G. Honig, R. I.Hume, The Journal of cell biology 103, 171 (July, 1986); M. G. Honig, R.I. Hume, Trends in neurosciences 12, 333 (September, 1989)) and fixed(G. E. Baker, B. E. Reese, Methods Cell Biol 38, 325 (1993); P.Godement, J. Vanselow, S. Thanos, F. Bonhoeffer, Development 101, 697(December, 1987)) tissues and cells. DiI labeling does not appreciablyaffect cell viability, development, or basic physiological properties.The dye uniformly labels neurons via lateral diffusion in the plasmamembrane. Coumarin Phalloidin (P2495, Sigma-Aldrich) was used to labelactin.

Secondary Antibodies:

The secondary antibodies used were Peroxidase AffiniPure Goat anti-mouse(Jackson ImmunoResearch, Suffolk, UK), Cy3-conjugated Goat Anti-Rat IgG,Cy5-conjugated goat anti-rabbit IgG, Rhodamine Red-X-AffiniPure FabFragment Goat Anti-Rabbit IgG (Jackson ImmunoResearch). DyLight488-labeled secondary goat anti-mouse IgG, DyLight 633-labeled secondarygoat anti-rabbit IgG (KPL, Gaithersburg, Md., USA).

Gene Knockdown—RNA Interference and Transfection

siRNA Oligos

Double-stranded RNA can initiate post transcriptional gene silencing inmammalian cell cultures via a mechanism known as RNA interference(RNAi). The sequence-specific degradation of homologous mRNA istriggered by 21-nucleotide RNA-duplexes termed short interfering RNA(siRNA). The homologous strand of the siRNA guides a multi-proteincomplex, RNA-induced silencing complex (RISC), to cleave target mRNA.The siRNA against rat MAPRE3 (NM_(—)001007656) was obtained as anOn-Target plus smart pool L-099082-01-0005-0010 (Dharmacon, ThermoFisher Scientific, Lafayette, Colo., USA). Dharmacon ON-TARGETplusNon-targeting siRNA (D-001810-01-05) was used as a negative control. Tocontrol transfection efficiency, a control siRNA Dharmacon siGLORISC-free siRNA (D-001600-01-05), was used.

Transfection

Transfections were performed using Lipofectamine RNAiMAX (Invitrogen).Transfection was carried out according to manufacturer's protocol. Forrat MAPRE3 knockdown, PC12 cells or neurons were plated in growth mediumwithout antibiotics at concentration of 10⁴ cells/0.32 cm² such thatthey will be 30-50% confluent at the time of transfection. To obtain thehighest transfection efficiency and low non-specific effects, siRNAtransfection was performed in the following optimized conditions: 50 nMfor neurons and 25 nM PC12 of each siRNA construct with 0.2 μlLipofectamine RNAiMAX (Invitrogen) was added per well of the 96-wellplate and scaled up for different 24 or 6-well plates, according to themanufacturer's instructions. The medium was changed one day aftertransfection. 48 and 72 hours later, cells were harvested to assay forgene knockdown at the RNA and protein levels.

Zinc Toxicity

On the day of the experiment, the growth medium was aspirated and freshmedium containing ZnCl₂ (400 μM; Sigma, Rehovot, Israel) with or withoutNAP was added to the cells. The cells were incubated for 4 hrs at 37° C.5% CO₂. Survival was measured using the MTS assay.

Cell Viability Assay—Metabolic Activity Measurements

Metabolic activity of cells was measured by the CellTiter 96® AqueousNon-Radioactive Cell Proliferation kit in accordance with themanufacturer's instructions (Promega). The assay uses a colorimetricmethod for determining the number of viable cells employing atetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS] and an electron-coupling reagent, phenazine methosulfate (PMS). MTSis bio-reduced by cells into a formazan product that is detected at 490nm. The conversion of MTS into the formazan form is accomplished bydehydrogenase enzymes found in metabolically active cells. Thus, thequantity of formazan product as measured by the amount of 490 nmabsorbance is directly proportional to the number of living cells inculture (A. H. Cory, T. C. Owen, J. A. Barltrop, J. G. Cory, CancerCommun 3, 207 (July, 1991)).

RNA Extraction

Total RNA from cells was extracted using the RNeasy Plus Mini Kit(Qiagen, Hilden, Germany) according to the manufacturer's protocol.Briefly, cells were lysed and homogenized and genomic DNA was removedusing gDNA Eliminator column. After genomic DNA removal, RNA waspurified using RNeasy spin columns. A 10 μl aliquot was taken from eachsample to examine the RNA content and quality, as indicated below. Therest of the samples were stored for further examination at −80° C.

RNA integrity was determined by fractionation on 1% agarose gel andstaining with ethidium bromide (Sigma). Two bands indicating 18S and 28Sribosomal RNA subunits should appear, while a smear without evidence forthe two ribosomal RNA bands is indicative for RNA degradation. RNAquantity and quality were analyzed by the Nanodrop ND-1000 UV-Visspectrophotometer (NanoDrop Technologies, Wilmington, Del., USA). Eachsample measurement was performed in duplicates.

Reverse Transcription and Quantitative Real-Time Polymerase ChainReaction (PCR)

Samples containing equal amount of total RNA were used to synthesizesingle-strand cDNA using SuperScript III Reverse Transcriptase (RT,Invitrogen) or High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems), with random hexamers according to the manufacturer'sinstructions. In each RT-PCR run, two negative controls were included:sterile water without RNA to rule out contamination in the reactioncomponents and total RNA without the RT enzyme to test for genomiccontamination. Real-Time polymerase chain reaction (PCR) was performedusing the SYBR® Green PCR Master Mix or Fast SYBR® Green PCR Master Mixand ABI PRISM 7900 or ABI StepOnePlus Sequence Detection Systemsinstrument and software (Applied Biosystems) accordingly using thedefault thermocycler program for all genes: 10 minutes of pre-incubationat 95° C. followed by 40 cycles of 15 seconds at 95° C. and 1 minute at60° C. The comparative Ct method was used for quantification oftranscripts. In short, the method compares the Ct value of target geneto a house-keeping gene in a single sample. Because it takes severalcycles for enough products to be readily detectable, the plot offluorescence versus cycle number exhibits a sigmoidal appearance. Atlater cycles, the reaction substrates become depleted, PCR product nolonger doubles, and the curve begins to flatten. The point on the curvein which the amount of fluorescence begins to increase rapidly, usuallya few standard deviations above the baseline, is termed the thresholdcycle (Ct value).

Real-Time PCR reactions were carried out in a total volume of 15 μl in a96-well plate (Applied Biosystems) containing 7.5 μl of X2 SYBR® GreenPCR Master Mix and ˜233 nM of each sense and antisense primers; for thereactions carried out with the StepOnePlus system the volume in eachwell, the type of plate and Master Mix were adjusted accordingly.Efficiencies of all primers used were calculated as a precursory stepusing the standard curve method, according to the equation: E(efficiency)=[10(−1/slope)−1]×100 and were near 100% for all primers.Product specificity was confirmed routinely by melting curve analysis.Sequence comparisons were performed with the BLAST software(www.ncbi.hlm.nih.gov/BLAST/).

Relative Quantitation (RQ)

Relative Quantitation (RQ) of gene expression using Comparative CT(AACt) determines the change in expression of a nucleic acid sequence(target, EB3) in a test sample (treated cells) relative to the samesequence in a calibrator sample (untreated control) (K. J. Livak, T. D.Schmittgen, Methods 25, 402 (December, 2001)). Fold-expression changesare calculated using the equation 2^(−ΔΔcT). Relative quantities of thetargets are normalized against the relative quantities of the endogenouscontrol (HPRT1). Gene Expression plots show the expression level orfold-difference of the target sample relative to the calibrator. Becausethe calibrator is compared to itself, the expression level for thecalibrator is always 1.

Relative Standard Curve Method

This method requires the least amount of validation because the PCRefficiencies of the target and endogenous control do not have to beequivalent. This method requires that each reaction plate containstandard curves. This approach gives highly accurate quantitativeresults because unknown sample quantitative values are interpolated fromthe standard curve(s). The relative standard curve method was used todetermine relative target (EB3) quantity in samples. With the relativestandard curve method, the software measures amplification of the target(EB3) and of the endogenous control (HPRT1) in samples, in a referencesample, and in a standard dilution series. Measurements are normalizedusing the endogenous control. Data from the standard dilution series areused to generate the standard curve. Using the standard curve, thesoftware interpolates target quantity in the samples and in thereference sample. The software determines the relative quantity oftarget in each sample by comparing target quantity in each sample totarget quantity in the reference sample.

Primers

Primer pairs (Table 1) were designed using the primer 3 web interface(http://frodo.wi.mit.edu/primer3/) or by using the NCBI primer designingtool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesizedby Sigma-Genosys. To avoid amplification of contaminating genomic DNA,primers for quantitative real time PCR were designed to target exon-exonjunction.

RNA expression levels were normalized to mouse/rat HPRT1(hypoxanthine-guanine phosphoribosyltransferase) as endogenous control.

TABLE 1 Quantitative Real-Time PCR: primer pairs Primer SequenceMouse MAPRE1 5′-GCGTTGACAAAATAATTCCT-3′ (NM_007896)5′-TGGCAGCTACAGGATCATAC-3′ Mouse MAPRE2 5′-ATACAGCTCAACGAGCAGGTACAT-3′(NM_001162941) 5′-CAGCAGCTCAATCTCTCTCAACTTC-3′ Mouse MAPRE35′-GCTGTGTTCACTTGAGGAAG-3′ (NM_133350) 5′-GAATGATTTTGTCAACACCC-3′Mouse HPRT1 5′-GGATTTGAATCACGTTTGTGTC-3′ (NM_013556)5′-CAGGACTCCTCGTATTTGCAG-3′ Rat MAPRE1 5′-GAAGAAAGTGAAATTCCAGG-3′(NM_138509) 5′-AGGAATTATTTTGTCAACGC-3′ Rat MAPRE25′-GGGCGTTTCCAAGACAACCT-3′ (NM_001101000) 5′-CTTGTCGAGCCTCAACAGGAT-3′Rat MAPRE3 5′-GGACAAAATCATTCCCGTAG-3′ (NM_001007656)5′-GGTTGTAATCCTTTCCATCA-3′ Rat HPRT1 5′-AGGCCAGACTITGTTGGATT-3′(NM_012583) 5′-GCTTTTCCACTTTCGCTGAT-3′

Protein Extraction and Quantification

Total protein (cytoplasmic and nuclear fractions) extraction wasperformed from cells grown in 6 well culture plates using modified RIPAlysis buffer. Cell pellet was collected by centrifugation (300 g, 5 min,RT), washed twice in ice-cold PBS and subjected to lysis in modifiedRIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EGTA, 1% TritonX-100, 0.1% SDS, 0.1% sodium Deoxycholate) supplemented with proteaseinhibitor cocktail (Sigma) and 5 mM ethylenediaminetetraacetic acid(EDTA). Cells in modified RIPA buffer (50-150 μl/well) were gentlydisrupted, using a pipette, and then rotated for 20 min at 4° C. at 600angle. The resulting homogenate was centrifuged (16,000 g, 15 min, 4°C.), and the supernatant was then collected in aliquots and stored at−80° C. Proteins were quantified using the BCA™ Protein Assay Kit(Pierce, Rockford, Ill., USA). This method is based on the reduction ofCu²⁺ to Cu¹⁺ by protein in an alkaline medium (the biuret reaction),that is coupled to the colorimetric detection of the cuprous cation(Cu¹⁺) by using a reagent containing bicinchoninic acid (BCA). Thepurple-colored reaction product of this assay is formed by the chelationof two molecules of BCA with one cuprous ion. The resultingwater-soluble complex exhibits a strong absorbance at 562 nm that isnearly linear with increasing protein concentrations over a proteinrange of 20 μg/ml to 2000 μg/ml (P. K. Smith et al., Anal Biochem 150,76 (October, 1985); K. J. Wiechelman, R. D. Braun, J. D. Fitzpatrick,Anal Biochem 175, 231 (Nov. 15, 1988)).

Immunoblotting

For western blot analysis, sample buffer X5 (125 mM Tris-HCl pH 6.8, 25%β-mercaptoethanol (Sigma), 43.5% glycerol, 10% SDS, 0.05% bromophenolblue) was added to protein samples that were further denatured byboiling at 100° C. for 5 minutes. ˜10-20 μg protein extract per lane wasseparated by electrophoresis on a 10% (w/v) polyacrylamide gel (BioRad,Hercules, Calif., USA) containing 0.1% SDS or 4-20% precast iGels(NuSep, Bogart, Ga., USA). Molecular weights were determined usingPrecision Plus Protein Standards (10-250kD, Dual Color, BioRad).Following electrophoresis, proteins were transferred to nitrocellulosemembrane (Whatman plc, Kent, UK) and nonspecific antigen sites wereblocked using a solution containing 5% non-fat dried milk (w/v) in TBST(10 mM Tris pH 8, 150 mM NaCl, and 0.05% Tween 20) for 1 hour at roomtemperature. Antigen detection was performed over-night at 4° C. usingappropriate antibodies. Antibody-antigen complexes were detected usinghorseradish peroxidase (HRP) conjugated goat anti-mouse or anti-rabbitIgG secondary antibodies (Jackson ImmunoResearch, West Grove, Pa., USA)and visualized by the Pierce ECL Western Blotting Substrate kit (Pierce)on Kodak Biomax ML Scientific imaging film (Kodak, Chalon-sur-Saône,France). The densitometric analysis of western blots was performed withMiniBIS Pro Gel imaging system and software (DNR, Mahale HaHamisha,Israel).

Recombinant EB3 Production

Recombinant EB3 was prepared as previously described (S. Honnappa etal., Cell 138, 366 (Jul. 23, 2009)). Full-length human EB3 was subclonedinto pEX-N-His vector (PrecisionShuttle bacterial expression vector withN-terminal His tag CW300309, OriGene, Rockville, Md., USA). Competent E.coli BL21(DE3) were transformed with the vector on LB agar platescontaining 30 mg/ml kanamycin and 100 mg/ml chloramphenicol. Affinitypurification of the N-terminal 6xHis-tagged fusion proteins byimmobilized metal affinity chromatography on Ni²⁺-Sepharose (Amersham)was performed at 4° C. according to the manufacturer's instructions. The6xHis fusion-tag was removed from recombinant proteins and peptides bythrombin (Sigma) cleavage. Protein and peptide samples were gel filteredon a Superdex-75 column (Amersham) equilibrated in 20 mM Tris-HCl (pH7.5), supplemented with 75 mM NaCl.

Sulfolink Coupling Gel-NAP Affinity Chromatography

The affinity chromatography column included SulfoLink® ImmobilizationKit for Peptides (44999, Thermo Scientific, Rockford, Ill., USA).Binding of CKKKGGNAPVSIPQ was performed according to the manufacturers'instruction. Recombinant protein was loaded (2 mg/mL) on the columns (2mL) and incubated overnight at 4° C.; the columns were then washed withphosphate-buffered saline until all unbound protein was eluted [asconfirmed by protein assay (Bradford; Bio-Rad Laboratories, Mannheim,Germany)]. NAP-binding proteins were eluted in IgG Elution buffer (pH2.8) (Thermo scientific). In order to show specificity, binding wasinhibited with either NAP or control peptides. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performedfollowed by western blot analysis in order to detect the protein (R.Zamostiano et al., J Biol Chem 276, 708 (Jan. 5, 2001)).

Statistical Analysis

Results were analyzed for statistical significance between two groups byStudent's t-test and for multiple comparisons by one way analysis ofvariance (ANOVA). Data are presented as the mean±SEM from at least 3independent experiments performed in triplicates in western blotanalysis and at least 3 independent experiments in duplicates or more inconfocal studies. Statistical analysis of the data was performed byusing one-way ANOVA with Dunnett's post-test using GraphPad Prismversion 5.00 (GraphPad Software, San Diego Calif. USA,www.graphpad.com), * P<0.05, ** P<0.01, *** P<0.001.

Results The EB Binding Groove, a Potential Bioinformatics Tool for DrugDesign:

As stated above, EB1 was shown to interact with a conserved binding sitein +TIPs—namely, SxIP. Using live cell experiments and in vitroreconstitution assays, it was demonstrated that a short polypeptidemotif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including thetumor suppressor APC, the transmembrane protein STIM1, and the kinesinMCAK, for localization to MT tips in an EB1-dependent manner. Highlyconserved C-terminal domain of EB1 recognizes this short linear sequencemotif found in a large number of important +TIPs for MT plus-endtracking. The most prominent contacts involve Ser, Ile, and Pro, whichoccupy the positions 1, 3, and 4 of the SxIP motif (S. Honnappa et al.,Cell 138, 366 (Jul. 23, 2009)).

The protein sequences of EB1, 2, and 3 were compared by multiplesequence alignment in order to assess the EB1 binding motif SxIP for EB2and EB3. The binding domain is conserved in rat, human and mouse,between the EBs. EB3 is slightly more similar to EB1 than EB2 to EB1.The main (SxIP) motif interacting residues are highly conserved betweenEB1, EB2, EB3, (FIG. 3, yellow rectangle), while the other residuesinvolved in the cavity which are likely to have interaction with theother residues of the binding peptide, are less conserved (FIG. 3, redrectangle). This analysis indicates an extensive homology in the SxIPbinding groove between the EB's, suggesting it may be also be a bindingmotif for EB2, and EB3. The differences between the sequences mayexplain the specificity of SxIP motif containing proteins with their EBpartner (FIG. 3).

TABLE 2 (part I). SxIP motif containing proteins involved in NAPactivity via interaction with EB3; additional + TIPS; and other proteinsof interest UniProt Gene Accession Name Name # Function Motif SRCN1 SRCkinase signaling Q9C0H9 Acts as a negative regulator of SIP inhibitor1/SNAP-25- SRC. Regulates dendritic spine interacting morphology.Involved in calcium- protein/p130Cas- dependent exocytosis. May play aassociated role in neurotransmitter release protein/p140Cap or synapsemaintenance. Binds EB3 APC2 Adenomatous O95996 Brain-specificadenomatous SSIP polyposis coli protein polyposis coli homologue.2/Adenomatous Promotes rapid degradation of polyposis coli protein-CTNNB1 and may function as a like tumor suppressor. May function in Wntsignaling. Binds EB3. APC Adenomatous P25054 Tumor suppressor.Participates in SQIP polyposis coli protein Wnt signaling as a negativeregulator. Acts as a mediator of ERBB2-dependent stabilization ofmicrotubules at the cell cortex. It is required for the localization ofMACF1 to the cell membrane and this localization of MACF1 is criticalfor its function in microtubule stabilization. MACF1 Microtubule-actinQ9UPN3 F-actin-binding protein which may SKIP cross-linking factor 1play a role in cross-linking actin to other cytoskeletal proteins andalso binds to microtubules. Plays an important role in ERBB2-dependentstabilization of microtubules at the cell cortex. Acts as a positiveregulator of Wnt receptor signaling pathway and is involved in thetranslocation of AXIN1 and its associated complex (composed of APC,CTNNB1 and GSK3B) from the cytoplasm to the cell membrane. Hasactin-regulated ATPase activity and is essential for controlling focaladhesions (FAs) assembly and dynamics. NAV1 Neuron navigator 1 Q8NEY1Associates with a subset of SGIP microtubule plus ends. Enriched inneuronal growth cones. May be involved in neuronal migration. NAV2Neuron navigator 2 Q8IVL1 Possesses 3′ to 5′ helicase activity SFIP andexonuclease activity. Involved in neuronal development, specifically inthe development of different sensory organs. NAV3 Neuron navigator 3Q8IVL0 May regulate IL2 production by T- SGIP cells. May be involved inneuron regeneration. Highly expressed in brain. (part II). Additional +TIPS: SXIP SxIP + TIPS Partners sequence Function Reference Plexin B1EB1 SGIP Neurite Laht et al., outgrowth BBA 2012 Plexin B3 EB1, EB2,SGIP Neurite Laht et al., EB3 outgrowth BBA 2012 hsMACF2 EB1 SKIP(microtubule actin crosslinking factor)? Similar to ACF7? The SxIPsequence not the same ACF7 EB1, Peripheral Mimori- CLASP 1, microtubuleKiyosue JCB CLASP 2 outgrowth & 2001; Zaoui stabilization et al, PNAS2010. CLASP1 EB1, CLIP- Stabilization Mimori- 170, CLIP- Kiyosue JCB115, ACF7 2005 CLASP2 EB1, CLIP- SKIP & Rescue Mimori- 170, CLIP- SRIPKiyosue JCB 115, ACF7 2005 hsAPC BE1, SQIP Cell polarity Barth et al.,(adenomatous MCAK and Semin Cell polyposis coli tumorigenesis Dev Biol.2008 hsSTIM1 EB1 TRIP ER remodeling Grigoriev et (stromal al., CurrBiol, interacting 2008 molecule) P140Cap EB3 Regulator of Jaworski etal., Src tyrosine Neuron 2009 kinase. EB3 regulates spine size bymodulating the turnover of p140Cap in spines. MCAK EB1, SKIP MTTanenbaum (KIF2c) Tip 150 depolymerase et al., BioArchi, 2011 Tip150EB1, ? Targets MCAK Jiang et al., MCAK toMT EMBO 2009 Navigators EB1,EB2, ? MT severing, Maes et al., EB3 MT transport Genomics, and 2002cytoskeleton organization Melanophilin EB1 SGIP Mitotic spindle Wu etal., positioning JCB 2005 CDK5RAP2 EB1 ? CDK5RAP2- Fong et al EB1complex Mol Biol Cell induces 2009 microtubule assembly RhoGEF2 EB1 ?GTP/GDP Rogers et al., (Dm) exchange, Curr Biol regulate 2004 dynamicsDDA (hs) aka EB1, EB3 SAIP Recruit Kif2a Jang et al., PSRC1 to themitotic JCB 2008; proline/serine- spindle and Hsieh et al., rich coiled-spindle poles-- Oncogene coil 1 depolymerizing 2007. (part III). Otherproteins of interest: UniProt Accession Gene Name Name # Function MotifMAP1S Microtubule- Q66K74 Microtubule-associated protein that SSIPassociated protein mediates aggregation of 1S/ Microtubule- mitochondriaresulting in cell death associated protein and genomic destruction(MAGD). 8 Plays a role in anchoring the microtubule-organizing center tothe centrosomes. MAP1LC3A Microtubule- Q9H492 Probably involved information of SKIP associated autophagosomal vacuoles proteins 1A/1B(autophago somes). light chain 3A MAP1A Microtubule- P78559 Structuralprotein involved in the SKIP associated protein filamentouscross-bridging between 1A microtubules and other skeletal elements.EB Expression Aligns with NAP Activity:

Reverse transcription and quantitative real time PCR analysis of mRNAexpression in rat non-differentiated PC12 cells, differentiated PC12cells treated with NGF and primary cultures of cortical astrocytes andneurons grown for 4 days in vitro (DIV) or 19DIV showed that EB3 ishighly enriched in cortical neurons. RNA silencing of EB3 in PC12 cellsand primary neuronal cultures compared to cells treated withnon-targeting siRNA resulted in up to 50% mRNA expression inhibition ofEB3 with no effect on the other EB's (FIG. 4 a). Further analysis ofmouse cell lines, showed that EB3 is preferentially expressed in mouseP19 cells subjected to neuro-glia differentiation by retinoic acid (RA)compared to non-differentiated or cardiac and skeletal muscledifferentiated P19 cells (DMSO). The mouse fibroblast line NIH 3T3 alsodid not show preferential expression of EB3 (FIG. 4 b). These resultsindicate that NAP-responsive cells (I. Gozes et al., J Mol Neurosci 20,315 (2003); I. Divinski, M. Holtser-Cochav, I. Vulih-Schultzman, R. A.Steingart, I. Gozes, J Neurochem 98, 973 (August, 2006)) preferentiallyexpress EB3, while cells that do not respond to NAP treatment likeNIH3T3 (I. Gozes et al., J Mol Neurosci 20, 315 (2003); I. Divinski, M.Holtser-Cochav, I. Vulih-Schultzman, R. A. Steingart, I. Gozes, JNeurochem 98, 973 (August, 2006)) express EB3 in a low basal nonpreferential way compared to EB1 and EB2, which are ubiquitouslyexpressed.

NAP-EB3 Binding on Column Chromatography:

The inventors previous studies showed that NAP interacts withbrain/neuron-glia specific tubulin (M. Holtser-Cochav, I. Divinski, I.Gozes, J Mol Neurosci 28, 303 (2006); I. Divinski, L. Mittelman, I.Gozes, J Biol Chem 279, 28531 (Jul. 2, 2004)). As indicated above, otherfindings suggested that the SIP moiety in NAPVSIPQ is essential for itsactivity (M. F. Wilkemeyer et al., Proc Natl Acad Sci USA 100, 8543(Jul. 8, 2003)). This current experiment was set out to expand theseprevious findings and to further explore NAP protein binding targets,aiming toward a better understanding on NAP mechanisms as an activepeptide. As EB3 is highly enriched in neurons and binds to MT, and EB1was shown to interact with other +TIPs through the binding motif SxIP(Honnappa et al., Cell 138, 366 (Jul. 23, 2009)) and comparativesequence analysis showed that EB3 sequence shares high similarity withthe SxIP motif binding cavity in EB1 as also verified by Laht et al.,(P. Laht, K. Pill, E. Haller, A. Veske, Biochim Biophys Acta, (Feb. 21,2012)), these experiments investigated if EB3 is a NAP binding protein.Full-length human EB3 recombinant protein was produced and NAPinteraction with the recombinant EB3 protein was tested using the columnchromatography method (I. Divinski, L. Mittelman, I. Gozes, J Biol Chem279, 28531 (Jul. 2, 2004)). 2 mg of recombinant full-length human EB3were exposed to 2 mg NAP sequences (CKKKGGNAPVSIPQ) covalently bound tosulfolink coupling gel. Column load, flow through, wash and elutionfractions were further analyzed by polyacrylamide gel electrophoresis,followed by western analysis with EB3 antibodies. FIG. 5 shows thewestern results, indicating that NAP associates with EB3. Further columnspecificity was shown by competition with NAPVSIPQ/EB3-mimetic peptides.Thus, the affinity column beads were incubated with recombinant EB3 and2 mg of either: i] soluble NAPVSIPQ (as positive control, which shoulddisplace all binding); ii] NAPVSKIPQ (representing an EB3-binding, NAPmimetic); iii] NAPVSAIPQ, a negative control—which does not contain theEB3-binding signature, and iv] NAPVAAAAQ, a negative control—which doesnot contain any of the EB3-binding signature. Column chromatography,elution and western blots were performed as above. The extract fractions(flow, wash and elution) were separated by electrophoresis followed byprotein detection (FIG. 6). Competition with NAPVSIPQ or NAPVSKIPQshowed no binding of EB3 to the column bound NAP (no EB3 in the elutedmaterial). On the other hand, no competition was apparent with NAPVSAIPQor NAPVAAAAQ, EB3 was bound to NAP and acid-eluted. In order to verifythat EB3 was indeed eluted from the column, beads were taken from thecolumn resin mixed with sample buffer and western blot analysis wasperformed (FIG. 6—“NAPVAAAAQ” most right lane). No EB3 immunoreactivitywas observed suggesting there was no EB3 protein bindingnon-specifically to the resin itself. These findings suggest EB3 to beNAP direct binding target protein through the SxIP binding motif.

Binding to Cellular Proteins to NAP (Affinity Chromatography with BrainExtracts)

Affinity chromatography was carried out as above with rat brain extractsas before (34, 36). However, several additional experiments wereundertaken and mass spectrometry was employed to identify additionalpotential NAP-interacting proteins that elute from the affinity column.

Results:

The experiment was repeated twice (including mass spectrometry) and theresults showed specific interaction of NAP with drebrin, that could beassociated with the NAP-EB interaction, as drebrin binds directly toEB3, see, e.g., Geraldo et al., Nature Cell Biology 10:1181-1189, 2008.Additional proteins that have been shown to bind NAP or D-SAL are listedin Table 3.

TABLE 3 Comparison between binding proteins of NAP and AL-309 (allD-amino acids SALLRSIPA - D-SAL), (D. E. Brenneman et al., J PharmacolExp Ther 309, 1190 (June, 2004)) on the respective affinity columns (twoindependent experiments): NAP1 D-SAL Molecular tubulin, beta 2 tubulin,beta [Mus Weight: tubulin, alpha 3c musculus] ~55,000 KD TubB2, B2c, B3,B5 tubulin, alpha 1B TubA1, A1C ~100 KD drebrin 1 [ 

 ] Specific NAP-binding (or D-SAL) is depicted in bold. Shared NAP andD-SAL binding are indicated.

Drebrin 1 [Rattus norvegicus]:

drebrin, located in the dendritic spines of the neuron, plays a role inthe synaptic plasticity together with actin filaments. Drebrin regulatesthe morphological changes of spines. It has been observed to besignificantly reduced in the brains of Alzheimer's and Down syndromepatients (K. S. Shim, G. Lubec, Neurosci Lett 324, 209 (May 24, 2002)),and has been reported to play a role during neuritogenesis through itsbinding with EB3 (Geraldo et al., Nature Cell Biology 10:1181-1189,2008).

NAP Protection Against Zinc Toxicity is Dependent on EB3:

NAP has been previously shown to protect against MT breakdown andtubulin aggregation in the presence of toxic concentration of zinc thatwere associated with neuronal (Divinski et al., Neurochem 98, 973(August, 2006)) and in glial cells death (Divinski et al., J Biol Chem279, 28531 (Jul. 2, 2004)). Here, in a pheochromocytoma (PC 12) cellsurvival assay, PC12 cells were exposed to increasing zinc concentrationfor 4 hrs. Significant cell death was observed at zincconcentration >200 μM (data not shown), corroborating previouslypublished data that established the EC50 for zinc cell killing effect at308±38 μM (Sanchez-Martin et al., Brain Res Bull 81, 458 (Mar. 16,2010)). The inventors chose to work with zinc concentrations that gaveconsistent and significant cell death, i.e., 400 μM zinc. NAP treatmentat concentrations of 10⁻¹⁵M and 10⁻⁹M showed a significant protectioneffect against cell death induced by zinc toxicity (FIG. 7 a). Furtherevaluation of the derivative peptides at concentration of 10⁻⁹M in thepresence of 400 μM zinc, showed that NAPVSKIPQ (denoted SKIP on thefigure) and Acetyl—NAPVSKIPQ-NH₂ (denoted Ac-SKIP on the figure)provided protection against zinc toxicity, mimicking NAP activity (FIG.7 a). NAPVSRIPQ and NAPVTRIPQ, which were used as controls (denoted SRIPand TRIP on the figure) were inactive (FIG. 7 b). FIG. 8 shows thatNAPVSIPQ does not protect against zinc intoxication, when EB3 issilenced.

NAP Effects MT in Dendritic Protrusions in Primary Neuronal Cells:

Most excitatory synapses in the mammalian brain are formed at tinydendritic protrusions, termed dendritic spines. A dendritic spine is asmall, club-like cell protrusion from neuronal dendrites that form thepostsynaptic component. Dynamic changes in spine structure are known tooccur during normal brain development, and are likely to contribute tosynaptic plasticity underlying processes such as learning and memory.These cell protrusions play a critical role in synaptic transmission andplasticity (K. Huang, A. El-Husseini, Curr Opin Neurobiol 15, 527(October, 2005)). MTs, long thought to be absent from dendritic spinesuntil very recently, are capable of controlling spine morphology (J.Jaworski et al., Neuron 61, 85 (Jan. 15, 2009)), contributing to thesynaptic plasticity at the dendritic spine level (J. Jaworski et al.,Neuron 61, 85 (Jan. 15, 2009); J. Gu, B. L. Firestein, J. Q. Zheng, JNeurosci 28, 12120 (Nov. 12, 2008); C. C. Hoogenraad, F. Bradke, Trendsin cell biology 19, 669 (December, 2009); X. Hu, C. Viesselmann, S. Nam,E. Merriam, E. W. Dent, J Neurosci 28, 13094 (Dec. 3, 2008); P. Penzes,D. P. Srivastava, K. M. Woolfrey, Neuron 61, 3 (Jan. 15, 2009)). Thecytoskeleton of dendritic spines is particularly important in theirsynaptic plasticity; without a dynamic cytoskeleton, spines would beunable to rapidly change their volumes or shapes in responses tostimuli. The cytoskeleton of dendritic spines is primarily made offilamentous actin (F-actin). Spiny dendritic protrusions can beclassified on the basis of spine lifetime and motility into one of threecategories: filopodia, protospines, or spines (M. E. Dailey, S. J.Smith, The Journal of neuroscience: the official journal of the Societyfor Neuroscience 16, 2983 (May 1, 1996)). Mature dendritic spines areformed from dynamic spine precursors (filopodia and protospines) thatbecome stabilized, or occasionally by direct extension from the dendriteshaft. The conversion of filopodia to protospines coincides with theformation of a postsynaptic density (PSD) containing PSD-95 (G. S.Marrs, S. H. Green, M. E. Dailey, Nat Neurosci 4, 1006 (October, 2001);L. Qin, G. S. Marrs, R. McKim, M. E. Dailey, J Comp Neurol 440, 284(Nov. 19, 2001)). Location of post synaptic density proteins change inrelation to spine type and maturation, conversion of filopodia to spinesinvolves assembly of a core PSD scaffold (time-course: ˜0.5-2 hr) andPSDs in developing spines (proto-spines) are highly dynamic: they canrapidly appear or disappear, as well as grow, shrink, move and possiblysplit and merge (G. S. Marrs, S. H. Green, M. E. Dailey, Nat Neurosci 4,1006 (October, 2001); L. Qin, G. S. Marrs, R. McKim, M. E. Dailey, JComp Neurol 440, 284 (Nov. 19, 2001); G. S. Mans et al., Mol CellNeurosci 32, 230 (July, 2006); S. J. Schachtele, J. Losh, M. E. Dailey,S. H. Green, J Comp Neurol 519, 3327 (Nov. 1, 2011)). Here, we askedwhether both dynamic (tyrosinated—Tyr-MT) and stable (de-tyrosinated,Glu-MT) MTs can be found in dendritic protrusions. PSD-95 was used as amarker for mature dendritic spines. Neurons were exposed to NAP for 2hrs. Using advanced techniques in confocal microscopy, both Tyr-MT andGlu-MT were shown to be present in dendritic protrusions either in thepresence of or in the absence of NAP (FIG. 9).

The inventors' recent findings suggest NAP control of the tubulintyrosination cycle (Oz and Gozes, 2012, JBC, invited revision). Hence,the fact that dynamic MT and stable MTs are found in the dendriticprotrusions suggests potential involvement in spine dynamics.

NAP Effect on PSD-95 Density in Primary Cortical Neuron Culture isEB3-Dependent:

Increased neuronal activity enhances both the number of spines invadedby MTs and the time that the MTs spend in the spines. EB3 knockdownsignificantly reduced the number of spines in cultured hippocampalneurons (X. Hu, C. Viesselmann, S. Nam, E. Merriam, E. W. Dent, JNeurosci 28, 13094 (Dec. 3, 2008)). Aβ oligomers, the hallmark ofAlzheimer's disease, bind to synaptic sites (P. N. Lacor et al., TheJournal of neuroscience: the official journal of the Society forNeuroscience 24, 10191 (Nov. 10, 2004)) and reduce the density of spinesin organotypic hippocampal slice cultures (H. Hsieh et al., Neuron 52,831 (Dec. 7, 2006); G. M. Shankar et al., The Journal of neuroscience:the official journal of the Society for Neuroscience 27, 2866 (Mar. 14,2007); B. R. Shrestha et al., Mol Cell Neurosci 33, 274 (November,2006); W. Wei et al., Nat Neurosci 13, 190 (February, 2010)),dissociated cultured neurons (B. Calabrese et al., Mol Cell Neurosci 35,183 (June, 2007); N. A. Evans et al., J Neurosci Methods 175, 96 (Oct.30, 2008); P. N. Lacor et al., The Journal of neuroscience: the officialjournal of the Society for Neuroscience 27, 796 (Jan. 24, 2007)) andtransgenic mouse models (T. A. Lanz, D. B. Carter, K. M. Merchant,Neurobiology of disease 13, 246 (August, 2003); J. S. Jacobsen et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 103, 5161 (Mar. 28, 2006); T. L. Spires et al., The Journal ofneuroscience: the official journal of the Society for Neuroscience 25,7278 (Aug. 3, 2005)). Given the previously observed effect of NAP onneurite outgrowth (M. Pascual, C. Guerri, J Neurochem 103, 557 (October,2007); S. Chen, M. E. Charness, Proc Natl Acad Sci USA, (Dec. 1, 2008);W. A. Lagreze et al., Invest Ophthalmol Vis Sci 46, 933 (March, 2005);V. L. Smith-Swintosky, I. Gozes, D. E. Brenneman, M. R. D'Andrea, C. R.Plata-Salaman, J Mol Neurosci 25, 225 (2005)), the MT involvement indendritic spine formation (J. Jaworski et al., Neuron 61, 85 (Jan. 15,2009); J. Gu, B. L. Firestein, J. Q. Zheng, J Neurosci 28, 12120 (Nov.12, 2008); C. C. Hoogenraad, F. Bradke, Trends in cell biology 19, 669(December, 2009); X. Hu, C. Viesselmann, S. Nam, E. Merriam, E. W. Dent,J Neurosci 28, 13094 (Dec. 3, 2008); P. Penzes, D. P. Srivastava, K. M.Woolfrey, Neuron 61, 3 (Jan. 15, 2009)), the inventors questioned if NAPaffects dendritic spine density in primary cortical neurons. To measuredendritic spine density, the post synaptic density protein, PSD-95, wasused. The presence of PSD-95 clusters in excitatory neurons is wellcorrelated with the number of mature dendritic spines (V. A. Alvarez, B.L. Sabatini, Annual review of neuroscience 30, 79 (2007); M. J. Kennedy,M. D. Ehlers, Annual review of neuroscience 29, 325 (2006)). Neuronswere exposed to increasing concentrations of NAP for 2 hours. Results(FIG. 10 a,b) indicate a NAP effect on the density of PSD-95, withrespect to the control, in a bell-shaped dose response curve with a highsignificant effect at a concentration range of 10⁻¹⁵M-10⁻⁶M NAP. Theobserved effect, reached a peak value of 50% over control at a NAPconcentration of 10-12M (FIG. 10 b). Peptides derived from hybrids ofNAPVSIPQ and +TIPs binding motifs that contain SxIP also possessneurotrophic and neuroprotective activity as follows below. Three novelpeptides were tested in addition to NAP as outlined in FIG. 10 c.

Results showed activity for NAPVSKIPQ that was paralleled to NAPVSIPQactivity. Importantly, NAPVSAIPQ and NAPVAAAAQ, which did not replaceEB3-NAPVSIPQ binding (FIG. 6), did not enhance PSD-95 staining. Theaffinity chromatography results showed association of NAP with EB3. Arecent manuscript has shown EB3 interaction with PSD-95 at the level ofthe dendritic modeling and plasticity (E. S. Sweet et al., J Neurosci31, 1038 (Jan. 19, 2011)). Thus, EB3 plus-end decorated MTs controlactin dynamics and regulate spine morphology and synaptic plasticity,through interaction with PSD-95, and NMDA receptor activation (L. C.Kapitein et al., J Neurosci 31, 8194 (Jun. 1, 2011)). Here the inventorsshow for the first time that silencing EB3 mRNA abolished NAP activity,implicating EB3 in the NAP-related neurotrophic effects (FIG. 10 d).

Similar findings were also seen below (FIG. 11): NAP enhances PSD-95expression in dendrites. Neuronal cultures were grown as above andstained with monoclonal anti-PSD-95) developed by DyLight 488-labeledsecondary goat anti-mouse IgG and counter stained with the nuclearstain—DAPI.

Discussion

Cooperation of Binding Proteins at the MT +TIPS:

Honnappa et al. (S. Honnappa et al., Cell 138, 366 (Jul. 23, 2009))showed that the affinity of individual +TIP-EB1 interactions is ratherweak (low micromolar range). Their data demonstrate that multiple SxIPmotifs, either within the same polypeptide chain or within differentpolypeptide chains, cooperate to increase the EB-dependent targetingefficiency.

EB-Drebrin-NAP:

The present inventors' original affinity column results (above) havesuggested NAP-drebrin interaction. Drebrin is one of the majorF-actin-binding proteins in neurons. Two isoforms of drebrin, E and A,are found in mammals, with drebrin A being the form specificallyexpressed in neurons. The biological functions of drebrin A have beenreported in various publications, see, e.g., Kobayashi et al., J CompNeurol 503(5):618, 2007; Bazellieres et al., J Cell Sci Advance OnlinePublication Jan. 24, 2012; Mizui et al., J Neurochem 109(2):611, 2009.

Specificity of Interaction:

Besides the NAP-like peptides described above that do not interact withEB3, other peptides that have been assayed include the results below(FIG. 14, inactive NAPVSIAQ (P7A) and NAPVAIPQ (S5A)). Importantly, whentested on astrocytes that have shown MT reorganization after NAPapplication (2 and 4 hours (I. Divinski, L. Mittelman, I. Gozes, J BiolChem 279, 28531 (Jul. 2, 2004)), the addition of NAPVSIAQ did not mimicNAP activity on MT organization after 4 hours of incubation (FIG. 14).

Tubulin Tyrosination and the Association with EB:

Recent findings by the present inventors suggest NAP control of thetubulin tyrosination cycle (Oz and Gozes (2012) The ADNP DerivedPeptide, NAP Modulates the Tubulin Pool: Implication for Neurotrophicand Neuroprotective Activities (Translated from eng) PLoS One7(12):e51458). Hence, the fact that dynamic MT and stable MTs are foundin the dendritic protrusions suggests potential involvement in spinedynamics. Tubulin tyrosination is associated with +TIP binding tomicrotubules (Weisbrich et al., Nature Structure & Molecular Biology14:959, 2007). Indeed, NAP affected spine dynamics as measured at thelevel of PSD-95, showing increase in PSD-95 puncta after 2 hrincubation. The conversion of filopodia to protospines coincides withthe formation of a core postsynaptic density (PSD) which -containsPSD-95 (time-course: ˜0.5-2 hr). PSDs in proto-spines are highlydynamic: they can rapidly appear or disappear, as well as grow, shrink,move and possibly split and merge. The location of post synaptic densityproteins changes in relation to spine type and maturation. See, e.g.,Marrs et al., Nature Neuroscience 2001. 4(10): p. 1006, 2001; Qin etal., The Journal of Comparative Neurology 440(3):284, 2001; Marrs etal., Molecular and Cellular Neurosciences 32(3):230, 2006; andSchachtele et al., The Journal of Comparative Neurology 519(16): 3327,2011. Interestingly, NAP effects on the puncta have been shownthroughout the cell, similar in other systems, not associated with NAPtreatment, e.g., Sweet et al. (J Neurosci 31:1038, 2011), togethersuggesting that SD-95 alters microtubule dynamics via an associationwith EB3.

Example 2 A New, Active 4-Amino Acid Peptide SKIP (SEQ ID NO:6) I. InVitro Protection Methods Cells

Rat pheochromocytoma cell (PC12, ATCC, Bethesda, Md., USA) were grown in10-cm tissue culture dishes (Corning). The base medium for this cellline was RPMI-1640 Medium (Invitrogen) supplemented with 10%heat-inactivated horse serum, 5% fetal bovine serum and solutioncontaining 100 U/ml penicillin together with 100 mg/ml streptomycin(Biological Industries, Beit Haemek, Israel). The cells were incubatedin 95% air; 5% CO2 in a humidified incubator at 37° C. The medium waschanged every 2 to 3 days. The cells were subcultured when cell densityreached 4×10⁶ cells/ml. The cells were split in a ratio of 1:4.

Zinc Intoxication

On the day before the experiment cells were to be harvested,re-suspended and seeded on tissue culture dishes. For cell viabilitymeasurements, cells were seeded on Poly-D-Lysine coated 96-well tissueculture dishes (Sigma-Aldrich) at a concentration of 3×10⁴ cells/well.

Cell Viability Measurements

The cell viability is measured by colorimetric method that determinesthe number of viable cells in culture. This method is based on thebio-reduction of the tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;MTS] by living cells into a colored formazan product. This reaction isoccurring in the presence of an electron coupling reagent (phenazineethosulfate; PES). The amount of formazan product is measured by ELISAplate reader at 490 nm and is directly proportional to the number ofliving cells in culture (CellTiter 96® AQueous One Solution CellProliferation Assay; Promega, Madison, Wis., USA).

Results The Effect of Zinc on Cell Death in Presence or Absence of SKIP(SEQ ID NO:6) in PC12 Cells

PC12 cells were exposed to ZnCl₂ in concentration of 400 μM in presenceor absence of increasing concentrations of SKIP (SEQ ID NO:6). ZnCl₂concentration of 400 RM was used based on previous results (Oz et al.2012 The ADNP Derived Peptide, NAP Modulates the Tubulin Pool:Implication for Neurotrophic and Neuroprotective Activities. PLoS One 7,e51458). Zinc treatment results in ˜20% cell death, which was protectedby the addition of SKIP, over a wide range of concentrations (FIGS. 15 Aand B). Another analogue that was tested, NAPVSGIPQ (SEQ ID NO:5), alsoshowed protection in the same PC12 cell/ZnCl₂ assays at theconcentration of 10⁻¹³M, reaching a level of statistical significance(FIG. 19).

II. In Vivo Protection Methods Animals

All procedures involving animals were approved by the Animal Care andUse Committee of Tel Aviv University and the National Institutes ofHealth (Bethesda, Md.). ADNP heterozygous mice, a model for cognitiveimpairments (Vulih-Shultzman et al., 2007, J. Pharmacol. Exp. Ther.323:438-449), were housed in a 12-h light/12-h dark cycle facility, andfree access to rodent chow and water was available.

SKIP (SEQ ID NO:6) Treatment

SKIP (SEQ ID NO:6) treatment included daily intranasal administrationsover a 1 month period to 5-month-old male mice (2 μg/5 μl/mouse/day).For intranasal administration, the peptide was dissolved in a vehiclesolution, in which each milliliter included 7.5 mg of NaCl, 1.7 mg ofcitric acid monohydrate, 3 mg of disodium phosphate dihydrate, and 0.2mg of benzalkonium chloride solution (50%). SKIP (SEQ ID NO:6) orvehicle solution (DD) was administered to mice hand-held in a semisupineposition with nostrils facing the investigator. A pipette tip was usedto administer 5 μl/each nostril. The mouse was handheld until thesolution was totally absorbed (˜10 s). Nasal SKIP (SEQ ID NO:6)application was performed daily, twice a day, for 1 month (5 days aweek). After 1 month, SKIP (SEQ ID NO:6) was applied 2 h before thebehavioral tests (described below).

Object Recognition Test

The test includes 2 consecutive days of habituation (five minutes perday) and the experimental day which consists of the three phases. Thetest was conducted two hours after the daily intranasal SKIP (SEQ IDNO:6). During the first phase (Phase 1, habituation phase), the openfield apparatus (arena of 50×50 cm) contained two identical objects(samples) and a mouse was placed in the apparatus facing the wall andallowed to freely explore the objects. At the end of the 5-min sessionof Phase 1, the mouse was put back into its home cage for 3 hours.Subsequently, the mouse was placed back into the apparatus for 3 min forthe second phase (Phase 2, short retention choice phase), during whichone of the familiar (sample (objects was replaced with a novel object.Approximately 24 h after the completion of Phase 1 test, the mouse wasplaced into the apparatus for 3 min for the third phase (Phase 3, longretention choice phase), during which one of the familiar objects wasreplaced with another novel object. The mouse was kept in its home cagebetween Phases 2 and 3. The objects (made of plastic or metal, 4×5 cm²)were washed and dried, and the apparatus was wiped clean before thestart of each session for each mouse. The positions of the familiar andnovel objects during Phases 2 and 3 were counterbalanced within andbetween groups to exclude the possibility of positional effects, butwere kept the same for a given animal. The time spent sniffing/touchingeach object was measured. The data was analyzed using the followingformula: D1=b−a, when ‘a’ designated the time of exploration of thefamiliar object and ‘b’ designated the time of exploration of the novelobject. The formula evaluates the discrimination capacity of the micebetween the novel object and the familiar object.

Morris Water Maze (MWM)

The apparatus was a pool with a diameter of 140 cm, filled with opaquewater (23-24° C.). An escape platform (12×12 cm²) was placed 0.5 cmbelow the water surface. Two daily tests, constituting two blocks oftrials, 90 s each, were performed for 5 consecutive days. The platformlocation and the animal starting point were held constant within eachpair of daily tests, but they were changed from day to day. The micewere allowed to stay on the platform for 20 s before and after eachtrial. The time taken for an animal to reach the platform (latency) wasrecorded. The daily improvement (in seconds to reach the hiddenplatform) for each animal (in comparison to the starting day) wascalculated. On the fifth day, a probe test was performed after thesecond daily trial as follows. The platform was removed from the mazeand the distance traveled and the time spent by the mice in the pool'squarter where the platform used to be, were recorded (for a maximalperiod of 90 s). Mouse behavior in the quarter that is most distant fromthe target quarter was also measured. All measurements were recorded asthe percentage of the total time spent or total path traveled in thepool. To determine the general mobility of the mice in the pool, theswimming behavior of each animal was monitored and the total path lengthand swim velocity were calculated. Monitoring was performed with the HVSvideo tracking system (HVS Image Ltd., Hampton, UK).

Elevated Plus-Maze

The maze consisted of two open arms (50 cm×10 cm) and two closed arms 50cm×10 cm×40 cm), with arms of each type opposite to each other. The mazewas elevated to a height of 50 cm from the floor. The experiment wasconducted in a dimly lit testing room. Mice were placed into the centreof the maze facing an open arm and were left free to explore it for 5min. The number of entries into the open and closed arms and the timespent in the open or closed arms were registered. The data were analyzedusing the following formula: D2=(b−a)/(b+a), when ‘a’ designated thetime spent in the open arm and ‘b’ designated the time spent in theclosed arm.

Results Object Recognition Test

In this novel-object test, object recognition was distinguished by theanimal spending more time exploring the novel object. Thisobject-recognition procedure takes advantage of an animal's tendency toapproach and explore novelty, in order to do so they must remember anddifferentiate the familiar from the novel object. Animal performance inthe object recognition memory task is presented in FIG. 15. ADNP+/− miceshowed reduced time spent on the novel object, demonstrating a deficitin working memory (FIG. 16A). Treatment with SKIP for 1 month (twice aday) improved the memory. Two-way ANOVA revealed significant effect ofgenotype (F(1, 27)=6.5, p=0.017), and treatment (F(1,27)=4.5, p=0.045)in terms of the total time spent exploring all objects across the 3phases of testing. Measurements of long term memory, 24 hours after thefirst exposure, revealed significant difference between ADNP-deficientmice and control mice (P<0.05) (FIG. 16B). Furthermore, SKIP (SEQ IDNO:6) treatment resulted in improvement in long term memory for theADNP-deficient mice, bringing them to the control levels (FIG. 16B).

Morris Water Maze

The test was performed to assess potential spatial learning and memorydeficits under the influence of the ADNP-deficient phenotype andpossible reversal by the ADNP-derived neuroprotective peptide, SKIP (SEQID NO:6). In the study, 4-month-old ADNP+/+ and ADNP+/− male mice weretreated by intranasal administration of either vehicle or SKIP (dailytreatments, twice a day, for 1 month). The treated mice were subjectedto a Morris water maze at the age of 5 month and continued to receiveSKIP (SEQ ID NO:6) during the 5 testing days of the water maze.Behavioral assessments were performed in a water maze by measurements ofthe time required to find a hidden platform. Two daily tests wereperformed over 5 testing days. The platform location and the animal'sstarting point were held constant within each pair of daily trials, butthe location of the platform and the animal's starting point werechanged every day. In the first daily test, indicative of referencememory, ADNP+/− male mice were impaired compared with control animals(FIG. 17A). Furthermore, although the ADNP+/+ mice learned the taskafter 3 testing days, ADNP+/− male mice did not learn the task (FIG.16A). SKIP (SEQ ID NO:6) treatment improved learning on the 2nd day,which was not apparent in the vehicle-treated ADNP+/− mice (FIG. 17A).The daily performance of each mouse was calculated as the difference[decrease in latency (seconds) to find the hidden platform, i.e.,improvement in performance] from performance of the same mouse on thefirst testing day. In the second daily test, on the 5th day of theMorris water maze testing there was a statistically significantdifference (p<0.05, two tailed t-test) between the control (designatedADNP+/+) mice and the ADNP-deficient (designated ADNP+/−) mice (FIG.17B). While ADNP+/− mice did not improve their performance on the 5thday in the second daily test, SKIP (SEQ ID NO:6) treatment resulted inimprovement in the behavior of the ADNP-deficient mice, bringing them tothe control levels (FIG. 17B).

Elevated Plus-Maze

To assess anxiety-related behavior we conducted an elevated plus mazetest. In this test, control group (designated ADNP+/+) spent more timein the closed arms compared with the time spent in the open arms (FIG.18). On the other hand, the ADNP-deficient group (designated ADNP+/−)spent more time in the open arms (FIG. 18). ANOVA test showed asignificant difference (P=0.003) of the time spent in the open andclosed arms between ADNP+/+ mice and ADNP+/− mice. The test revealsrisky behavior of the ADNP-deficient mice as opposed to controlbehavior. SKIP (SEQ ID NO:6) treatment resulted in reduction in therisky behavior of the ADNP-deficient mice.

CONCLUSIONS

SKIP (SEQ ID NO:6), a 4-amino acid peptide provides neuroprotection invitro and cognitive protection in vivo following nasal administration.SKIP (SEQ ID NO:6) is the EB1/EB3 binding site, indicatingneuroprotection through EB/microtubule interaction. Peptides NAPVSIPQand NAPVSKIPQ have been shown to bind EB3 in vitro in a columnchromatography method described in Example 1. Since SKIP (SEQ ID NO:6)is a small peptide that can be readily made with high bioavailability,this peptide, including its modified variants (such as all D-amino acidSKIP and acetylated and/or amidated SKIP, e.g., acetyl-SKIP-NH₂) is ofsignificant potential value as a therapeutic agent for neuroprotection.

Example 3 Compounds that have been Identified as Modulators of CellDeath/Survival/Plasticity Based on their Interaction with EB3 or EB1Protein

TABLE 4 name Description TRAZODONE major depressive Anti-anxiety Agentsdisorders Antidepressants, Second-Generation Serotonin Uptake InhibitorsAntidepressive Agents, Second-Generation ACETOPHENAZINE It is used inthe Acetophenazine blocks postsynaptic mesolimbic dopaminergic treatmentof D1 and D2 receptors in the brain; disorganized and depresses therelease of psychotic thinking. It hypothalamic and hypophyseal is alsoused to help hormones and is believed to depress treat false thereticular activating system thus perceptions (e.g. affecting basalmetabolism, body hallucinations or temperature, wakefulness, delusions).It vasomotor tone, and emesis. primarily targets the dopamine D2receptor. Carphenazine treatment of acute or Carphenazine blockspostsynaptic mesolimbic dopaminergic chronic D1 and D2 receptors in thebrain; depresses the release of schizophrenic hypothalamic andhypophyseal hormones and is believed to reactions depress the reticularactivating system thus affecting basal metabolism, body temperature,wakefulness, vasomotor tone, and emesis. Flumazenil Fumazenil is anFlumazenil, an imidazobenzodiazepine derivative, antagonizesimidazobenzodiazepine the actions of benzodiazepines on the centralnervous system. derivative and a Flumazenil competitively inhibits theactivity at the potent benzodiazepine recognition site on theGABA/benzodiazepine benzodiazepine receptor complex. Flumazenil is aweak partial agonist in some receptor antagonist animal models ofactivity, but has little or no agonist activity in that competitivelyman. inhibits the activity at the benzodiazepine recognition site on theGABA/benzodiazepine receptor complex, thereby reversing the effects ofbenzodiazepine on the central nervous system. QUETIAPINE Quetiapine isQuetiapine's antipsychotic activity is likely due to a indicated for thecombination of antagonism at D2 receptors in the mesolimbic treatment ofpathway and 5HT2A receptors in the frontal cortex. schizophrenia asAntagonism at D2 receptors relieves positive symptoms while well as forthe antagonism at 5HT2A receptors relieves negative symptoms treatmentof acute of schizophrenia manic episodes associated with bipolar Idisorder RISPERIDONE antipsychotic drug Blockade of dopaminergic D2receptors in the limbic system with high affinity for alleviatespositive symptoms of schizophrenia such as 5-hydrotryptaminehallucinations, delusions, and erratic behavior and speech. (5-HT) andBlockade of serotonergic 5-HT₂ receptors in the mesocortical dopamine D2tract, receptors. It is used primarily in the management ofschizophrenia, inappropriate behavior in severe dementia and manicepisodes associated with bipolar I disorder. FLUVOXAMINE Fluvoxamine isan inhibition ofCNS neuronal uptake of serotonin. antidepressant whichfunctions pharmacologically as a selective serotonin reuptake inhibitor.Though it is in the same class as other SSRI drugs, it is most oftenused to treat obsessive- compulsive disorder. THIOTHIXENE A thioxanthineused Thiothixene acts as an antagonist (blocking agent) on different asan antipsychotic postsysnaptic receptors -on dopaminergic-receptors(subtypes agent D1, D2, D3 and D4 - different antipsychotic propertieson productive and unproductive symptoms), on serotonergic- receptors(5-HT1 and 5-HT2 DILTIAZEM vasodilating action Possibly by deforming thechannel, inhibiting ion-control gating due to its mechanisms, and/orinterfering with the release of calcium antagonism of the from thesarcoplasmic reticulum, diltiazem, like verapamil, actions of theinhibits the influx of extracellular calcium across both the calcium ionin myocardial and vascular smooth muscle cell membranes membranefunctions. It is also teratogenic ALMOTRIPTAN Almotriptan is aAlmotriptan binds with high affinity to human 5-HT_(1B) and 5- triptandrug for the HT_(1D) receptors leading to cranial blood vesselconstriction. treatment of migraine headaches METHYSERGIDE Methysergideis used prophylactically in migraine and other vascular headaches and toantagonize serotonin in the carcinoid syndrome

All patents, patent applications, and other publications, includingGenBank Accession Numbers, cited in this application are incorporated byreference in the entirety for all purposes.

INFORMAL SEQUENCE LISTING SEQ ID NO: 1 NAPVSxIPQ(x is one amino acid of any identity) SEQ ID NO: 2 NAPVSKIPQSEQ ID NO: 3 R¹-NAPVSxIPQ-R² SEQ ID NO: 4 R³-NAPVTxIPQ-R⁴(each of R¹, R², R³, and R⁴ is an amino acidsequence independent of each other, having atleast one, up to 40 amino acids of any identity) SEQ ID NO: 5 NAPVSGIPQSEQ ID NO: 6 SKIP SEQ ID NO: 7 SGIP SEQ ID NO: 8 SRIP

1. An isolated peptide, which (1) comprises a core amino acid sequenceof (a) SKIP (SEQ ID NO:6); (b) SGIP (SEQ ID NO:7); (c) SRIP (SEQ IDNO:8); or (d) NAPVSxIPQ (SEQ ID NO:1) or a conservatively modifiedvariant thereof; (2) has up to 40 amino acids at either or both of theN-terminus and the C-terminus of the core amino acid sequence; and (3)inhibits cell death or increases cell plasticity.
 2. The peptide ofclaim 1, wherein the core amino acid sequence is NAPVSKIPQ (SEQ ID NO:2)or NAPVSGIPQ (SEQ ID NO:5).
 3. The peptide of claim 1, which has up to20 amino acids at either or both of the N-terminus and the C-terminus.4. The peptide of claim 1, wherein the peptide consists of the coreamino acid sequence.
 5. The peptide of claim 1, wherein the peptideconsists of SKIP (SEQ ID NO:6).
 6. The peptide of claim 1, wherein thepeptide consists of SEQ ID NO:2 or SEQ ID NO:5.
 7. The peptide of claim1, wherein the peptide is acetylated at the N-terminus or at an internalK residue, or is lipidated at the N-terminus, or is amidated at theC-terminus.
 8. The peptide of claim 7, wherein the peptide isacetyl-SKIP-NH₂.
 9. The peptide of claim 1, wherein the core amino acidsequence comprises at least one D-amino acid.
 10. The peptide of claim9, wherein the peptide is all D-amino acid SKIP.
 11. The peptide ofclaim 1, wherein the cell is a neuronal cell.
 12. A method for promotingcell survival or plasticity, comprising contacting a cell with acompound in an amount that is effective to increase the expression of anEB protein.
 13. The method of claim 12, wherein the EB protein is an EB1protein, or an EB2 protein, or an EB3 protein.
 14. The method of claim12, wherein the cell is a neuronal cell.
 15. The method of claim 12,wherein the cell is present in a patient's body.
 16. The method of claim12, wherein the compound is a peptide comprising the amino acid sequenceof SKIP, SEQ ID NO:1, 2, or 5 and having up to 40 amino acids at eitheror both of the N-terminus and the C-terminus.
 17. The method of claim12, wherein the compound is a peptide consisting of the amino acidsequence of SKIP, SEQ ID NO:1, 2, or 5, an all D-amino acid SKIP oracetyl-SKIP-NH2 or a compound named in Table
 4. 18. The method of claim12, for treating cognitive impairment.
 19. A method for identifying amodulator of cell survival or plasticity, comprising: (1) contacting,under conditions permissible for protein-modulator binding, an EBprotein, with a candidate compound; (2) detecting binding between the EBprotein and the candidate compound; and (3) identifying the candidatecompound as a modulator of cell survival or plasticity when bindingbetween the EB protein and the candidate compound is detected.
 20. Themethod of claim 19, wherein step (1) further comprises providing adrebrin protein to interact with the EB protein and the candidatecompound.
 21. The method for identifying a modulator of cell survival orplasticity, comprising: (1) contacting a cell that expresses an EBprotein, under conditions permissible for the expression of the EBprotein, with a candidate compound; (2) detecting the expression levelof the EB protein in the cell; and (3) identifying the candidatecompound as a modulator that promotes cell survival or plasticity whenan increased expression level of the EB protein in the cell is detected,and identifying the candidate compound as a modulator that suppressescell survival or plasticity when a decreased expression level of the EBprotein in the cell is detected.
 22. The method of claim 21, whereinstep (1) further comprises providing a drebrin protein to interact withthe EB protein and the candidate compound.
 23. A method for inhibitingdeath or promoting survival or plasticity of a cell, comprisingcontacting the cell with an effective amount of a modulator thatpromotes cell survival or plasticity identified by the method of claim19.
 24. A method for inhibiting death or promoting survival orplasticity of a cell, comprising contacting the cell with an effectiveamount of a modulator that promotes cell survival or plasticityidentified by the method of claim
 21. 25. (canceled)
 26. (canceled) 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)32. (canceled)